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Di-iron-tyrosyl radical ribonucleotide reductases
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Di-iron-tyrosyl radical ribonucleotide reductases JoAnne Stubbe New insights have been gained into the formation of the di-iron tyrosyl radical cofactor, which is essential for nucleotide reduction in the class I ribonucleotide reductases. Recent advances include Insight into the function of the tyrosyl radical in initiation of nucleotide reduction. Address Chemistry and Biology Departments, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Current Opinion in Chemical Biology 2003, 7:183–188 This review comes from a themed section on Bioinorganic chemistry Edited by Joan Broderick and Dimitri Coucouvanis 1367-5931/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1367-5931(03)00025-5
Abbreviations RNR ribonucleotide reductase Y tyrosyl radical
Introduction Escherichia coli ribonucleotide reductase (RNR) is composed of a 1:1 complex of two homodimeric subunits: R1 and R2. R1 is the ‘business end’ of the molecule where nucleotides are converted to deoxynucleotides. R1’s active site houses the cysteines essential for catalysis and contains multiple binding sites for deoxynucleotides and ATP that govern specificity of nucleotide reduction and the rate of reduction. R2 houses the di-iron tyrosyl radical (Y) cofactor, which is essential for initiation of the nucleotide reduction process on R1. Seminal EPR experiments in conjunction with isotopic labeling methods identified this first known protein radical as a Y in E. coli R2 and established that it was essential for catalysis [1,2]. Since then, Ys or modified Ys have been shown to play essential roles in manganese-cluster-mediated O2 evolution from water in the photosynthetic reaction center II, in prostaglandin synthase, and in galactose oxidase. They also have been postulated to play a role in cytochrome oxidase and several heme-dependent peroxidases [3]. This review focuses on the di-iron-Y RNR and provides an update on our understanding of the biosynthesis in vitro and in vivo of the cofactor and its biological function.
Diferric-Y cofactor assembly in E. coli in vitro The first detailed mechanism of formation of the di-iron Y cofactor in RNR was proposed in 1991 on the basis of www.current-opinion.com
time-resolved physical biochemical methods that allowed monitoring of changes in the oxidation state of the irons during the course of the reaction (Figure 1) [4]. Highresolution structures of R2 in the diferrous form [5], the diferric form [6] and, more recently, in the presence of endogenous anions (such as azide), active site mutations (D84E [7] and double mutant Y122F/F208A [8]), and alternative metals (Mn, Co) [9,10] have played an essential role in proposing structures for intermediates along the pathway (Figure 2). These structures have demonstrated the flexibility of carboxylates E238, D84 and E204 (E. coli numbering) within the active site environment, consistent with the importance of carboxylate shifts in the chemistry of di-iron proteins [11]. Recent studies have filled in many of the missing details of the di-iron cofactor assembly mechanism. The stoichiometry of cluster assembly requires two irons and a reducing equivalent to carry out a four-electron reduction of O2 to H2O [12]. The fourth electron is supplied by tyrosine, Y122 in E. coli RNR. Recent kinetic and thermodynamic studies have suggested that one Fe binds rapidly (proposed to be Fe2, Figure 2) and tightly to apo R2 and that a slow (10 s1) conformational change limits the rate of binding of the second iron (Fe1), which binds with fivefold lower affinity [13,14]. This slow step can be bypassed by starting with R2 that has been pre-loaded with ferrous iron [15]. When the loaded diferrous R2 is mixed with O2, the first wellcharacterized intermediate detected depends on the availability of the reducing equivalent required for diiron Y formation. When the reducing equivalent is not available, an exchange-coupled intermediate composed of a tryptophan cation radical (W48Hþ) and a Fe3þ/Fe4þ di-iron cluster designated X is observed [16,17]. This W48Hþ intermediate, postulated on the basis of its absorption spectrum and the structural similarity of R2 to that in cytochrome c peroxidase [18] has recently been characterized in detail by EPR and Mo¨ssbauer methods [19]. These studies substantiated and extended the original model that either W48Hþ or X can oxidize Y122 to the Y under conditions where the reductant is limiting. Furthermore, convincing evidence was presented that the species with the absorption transient at 560 nm (putative W48Hþ) is EPR active and coupled to a second paramagnetic species X. The proposed structure for X remains unresolved, although a variety of physical methods have limited the possibilities. Recent structural studies have demonstrated that E238 is flexible and can coordinate with both Fe1 and Fe2 in a m-n1,n2 fashion [8]. While the direct relevance of these structures to the coordination of the irons during catalysis of cluster assembly is unclear, the short Fe1 to Fe2 distance (2.5 A˚) in X requires a similar coordination of at least one of the Current Opinion in Chemical Biology 2003, 7:183–188
184 Bioinorganic chemistry
Figure 1
Y122 apoR2 + Fe2+ W48
Y122 apoR2 Fe2+ W48
Fast
Y122 Fe2+ Fe2+ W48
Y122
Fe2+ Y122 Fast R2 Fe2+ W48
10 s−1
?
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=X
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bridging carboxylates [20]. Thus, the crystallographic data suggest that a (m-n1,n2) E238 coordination is possible in X. Recent studies have shown the presence of intermediate X and its kinetic competence in assembly of the di-iron cofactor in mouse R2 [21], in contrast with earlier studies [22]. Thus, a congruence between eukaryotic and prokaryotic cluster assembly has been established.
O
Fe3+ W48 Native R2 3+
Fe
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Proposed mechanism for the assembly of the diferric-Y Cofactor of R2. The model is an expanded version of the original model based on recent time-resolved physical biochemical methods [14,19,23].
New insight into cluster assembly before formation of X has recently been provided. In the X-ray structure of reduced R2, both irons are four-coordinate, 3.9 A˚ apart, and E238 and E115 bridge the two irons in a m-1,3 configuration. Recent circular dichroism. magnetic circular dichroism and variable temperature, variable field magnetic circular dichroism experiments suggest, however, that Fe1 is four-coordinate and Fe2 is five-coordinate in solution, with E204 being bidentate to Fe2 [13]. A comparison of the coordination of these irons with the two irons in delta-9-desaturase and their differences in chemical reactivity with O2, suggests that a 4-coordinate/ 5-coordinate diferrous center enhances reactivity toward O2. Studies with the double mutant F208A and Y122F-R2 soaked with azide, an O2 surrogate, suggest that Fe2 is the site of O2 binding [5]. Additional experiments in which D84 is replaced with glutamic acid have provided insight into early stages of cluster assembly. Incubation of the diferrous form of the
Figure 2
(a)
(b)
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Demonstration of the flexibility of E238, D84. A comparison of (a) structures of wt R2 in the oxidized state [18] and (b) the reduced state [5] with (c) the D84E-R2 in the reduced state [7] and (d) the F208A, Y122F-R2 in the reduced state soaked with azide [8]. Current Opinion in Chemical Biology 2003, 7:183–188
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Di-iron-tyrosyl radical ribonucleotide reductases Stubbe 185
mutant with O2 generated a peroxide intermediate postulated to be on the reaction pathway in cluster assembly in wt-R2 [23]. The peroxide in the D84E-R2 has been characterized by resonance Raman [24], Mo¨ ssbauer and visible spectroscopies. Its isomer shift and visible spectrum is similar to a transient observed in wt-R2 cluster assembly [15]. Unfortunately, in the wt case, the species accounted for <10% of R2 and disappeared within 10 ms. A kinetics experiment in which the reaction is triggered with light is essential to determine if this species in wt-R2 is on the pathway and if it is structurally similar to peroxide observed with D84E-R2.
Yeast RNR: a system to examine cluster assembly in vivo Early stages of cluster assembly in R2s have been difficult to study because of inability in vitro to deliver Fe2þ and the extra reducing equivalent in a controlled fashion. Presumably, proteins play this role in vivo. Yeast is an excellent system to study cluster assembly in vitro and in vivo. Many of the genes involved in iron and copper homeostasis have been identified [25]. Genome sequences and isogenic strains of yeast with each gene deleted are available and genes for two RNRs, involved in replication and repair, have been identified [26,27]. The R1s of yeast RNR have been designated Y1 and Y3 and the R2s have been designated Y2 and Y4. Y4 is of particular interest because sequence comparison with Y2 reveals that it lacks three of the conserved iron ligands found in almost all other R2 sequences. In Y4, H118 and H241 (E. coli numbering) are each replaced with tyrosine and E238 is replaced with arginine (Figure 2) [28,29]. The arginine substitution precludes iron binding to the Fe2 site. Our hypothesis for the role of Y4 [30,31] was based on the seminal experiments in yeast, studying assembly of copper cofactors in proteins. A copper requiring protein such as superoxide dismutase has its copper delivered by a copper chaperone protein (Lys7) that is structurally homologous to superoxide dismutase [32]. Furthermore, the chaperone protein has its active site ligands modified relative to the receptor protein so that copper cannot bind. However, it also contains additional small domains that bind and deliver the copper [33,34]. The mechanism(s) for this delivery are presently under intense investigation. Our initial model proposed Y4 as a chaperone protein (Lys7 equivalent) that could deliver Fe2þ to Y2. Y4 was shown to be essential for di-iron-Y formation [30]. Incubation of Y2 and Y4 homodimers at physiological concentrations with Fe2þ and O2 in vitro resulted in rapid reorganization to generate a heterodimer Y2Y4 (one monomer of Y2 and one of Y4). Y2Y4 contained 0.6Y and 1.8 irons [35]. In vitro, the heterodimer is active with Y1 in nucleotide reduction. The structure of the heterodimer has recently been solved [36]. The structure of Y4 has also been solved to 3.1 A˚ and the structure of Y2 to 3.15 A˚ is undergoing final refinement (AC Rosenzweig et al., unpublished results). Whether Y2Y4 can re-equiliwww.current-opinion.com
brate and generate a Y2 homodimer with an active cofactor is an important unresolved issue. Studies from Thelander and co-workers [37], independently proposed that the heterodimer of Y2Y4 is active based on isolation of the Y2Y4 heterodimer when the genes for Y2 and Y4 were co-transformed in E. coli and the resulting R2 was isolated. They proposed that Y4 is a chaperone protein that prevents Y2 unfolding. Problems with both models are apparent, however, as several different yeast strains in which Y4 has been deleted are viable. While the DY4 strains are growth-compromised, their ability to grow raises the question as to the actual role of Y4 in vivo. We have recently demonstrated that one Y4 deletion strain has dramatically overproduced Y2. This strain may prove useful in purifying the factors required for cluster assembly. In E. coli, 1.2 Ys per R2 homodimer have been observed in many laboratories. In yeast, 0.6Ys per Y2Y4 heterodimer has been reported. The stoichiometry raises the possibility of half-sites reactivity of R2, which would require the R1R2 complex, essential for the nucleotide reduction process, to be unsymmetrical. In yeast, if Y2Y4 is the active species in vivo, then half-sites activity is required. Use of epitope-tagged R2s integrated into the genome under control of the wt-promoter should allow rapid removal of R2 from crude extracts and reveal if there are one or two Ys per R2. The biosynthesis of dinuclear non-heme iron systems still remains to be unraveled and the genetics and biochemistry accessible in yeast provides an excellent system to investigate this pathway. Unraveling this pathway should lead to a better understanding of the mechanism of Fe2þ binding and O2 reduction in vitro and iron deliver in vivo. Determination of the number of Ys per R2 in vivo is also essential for understanding R1R2 interactions and the details of the reduction process.
Function of Y in ribonucleotide reduction Seminal experiments in the 1970s demonstrated that Y122 of E. coli R2 is essential for the nucleotide reduction process [38]. Recently, its role in this process has been elucidated by a comparison of the structures of the three classes of RNRs [39,40,41]. RNR classifications are based on the different metallo-cofactors that initiate nucleotide reduction. The class II RNR uses adenosylcobalamin and the class III RNR utilizes a glycyl radical (G) generated by a 4Fe4S cluster and S-adenosylmethionine [42]. A comparison of the class II structure with the class I and III structures substantiated a common active site architecture where nucleotide reduction occurs. However, the structural comparison unexpectedly revealed that the location of the radical initiators in the class II and III RNRs overlapped with two conserved Ys (Y731 and Y730) in the C-terminus of R1 in the class I RNRs [41]. The mechanism of radical initiation in the Current Opinion in Chemical Biology 2003, 7:183–188
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Figure 3
Figure 4
E441 GDP
3.4 Å C439
R2 peptide (375)
Y730
3.4 Å 3.3 Å
R2 peptide (360) R2 (340)
Y731
R2 peptide (360) R2 (340)
25 Å Current Opinion in Chemical Biology
R1 Y356?
R2 A Docking model of R1 and R2 adapted from the original model of Uhlin and Eklund [45]. In this model, R1 (a homodimer) is complementary in shape to R2 (a homodimer). In R2, the diiron center is shown as red balls. The cyan colored residues in stick format are amino acid residues proposed to play an important role in proton coupled electron transfer between R2 and R1. The C-terminus of each monomer of R2 (340) is indicated. The C-terminal 35 amino acids important in binding to R1 are disordered. Y356-R2 is a conserved residue found in this region. R1 has space-filling renditions of the substrate and effectors as indicated. The cyan-colored residues in stick format are amino acid residues proposed to play an important role in proton-coupled electron transfer between R2 and R1. The R2 peptide (360–375 in red) required for R1 to crystallize is shown.
3.1 Å 7.6 Å
The radical initiation in the class I RNRs is obviously more complex than in the other classes of RNRs. A docking model of R1 and R2 provides the working hypothesis for radical initiation: how the Y122 on R2 generates a S on R1 which is 35 A˚ distant (Figure 3) [46]. The structural model is based on the complementary shapes of R2 and R1 and the observation that the C-terminal 10–20 amino acids of R2s (disordered in all structures) are essential for binding R2 to R1 [47]. Additionally, the crystallization of R1 required the presence of the C-terminal peptide of R2, 15 residues (360–375) of which are apparent in the R1 structure [45]. The structural model, conserved residues from sequence alignments, and the long distance between Y122 on R2 and C439 on R1 suggests that the radical initiation involves a pathway with transient amino radical intermediates Current Opinion in Chemical Biology 2003, 7:183–188
Fe2 4.2 Å
Y122
class II RNR has been shown to involve hydrogen atom abstraction to generate the transient S initiator [43]. Based on the structure and chemistry, the G in the class III RNRs probably generates a putative S by hydrogen atom abstraction as well [44]. By analogy, therefore, the two Ys in the class I RNRs may be involved in hydrogen atom abstraction to generate the putative active site S [45].
D237
W48
Fe1 Current Opinion in Chemical Biology
A model for the proposed radical initiation pathway. All residues indicated are conserved. The position of Y356 relative to W48 in R2 and Y731 in R1 is unknown as it is located on the disordered C-terminus of R2 ((340) Figure 3).
(Figure 4) [48]. The rate constant for nucleotide reduction, 2–10 s1, and Marcus theory for electron transfer, require intermediates in S formation, given the long distance. Several constraints on the radical initiation process have made the mechanism difficult to study. Y is oxidized and reduced on each turnover (J Ge, personal communication) and the Y hole in R2 is the stable form of RNR. Oxidation of a cysteine by a Y is thermodynamically uphill, requiring cysteine deprotonation [42]. Cysteine oxidation is coupled to a rapid irreversible dehydration step in nucleotide reduction to facilitate the uphill process. Thus, concentrations of intermediates are expected to be low and difficult to detect. The rate-determining step in the nucleotide reduction process is a physical step gated by binding of allosteric effector and binding of substrate to R1 (J Ge, personal communication). This conformational change is www.current-opinion.com
Di-iron-tyrosyl radical ribonucleotide reductases Stubbe 187
followed by rapid electron transfer and rapid nucleotide reduction. Thus, to detect amino acid radical intermediates, the rate determining step must be changed or a trap must be placed within the pathway. The radical initiation process probably involves both tunneling and hopping mechanisms [49]. A tunneling mechanism between Y122 and W48 involving electron transfer and protonation is proposed and is based on studies of diiron Y cluster assembly on R2. As noted above, the transient W48þ can oxidize Y122 to Y122 [17,19]. The reversibility of this process hinges on the coupling of oxidation potential with protonation state of the amino acids [50] and suggests the importance of the proton transfer between W48 and D237. The inactivity of D237N-R2 [51] and the low activity of D237E-R2 support this proposal. No structural information is available about the distance between Y356 (R2) and W48 in R2 or Y731 in R1. The docking model suggests distances of 12 A˚ , well within the constraints of Marcus theory for rapid electron transfer. Mutagenesis studies have demonstrated that the conserved residues in this pathway (Figure 4) are important [51,52]. Finally, the inactivity of Y730F and Y731FR1s in the nucleotide reduction process, implicate a hydrogen atom transfer mechanism in this part of the radical initiation pathway. No amino acid radical intermediates have been detected, as required by this model. Thus, although Y122 in R2 is essential for nucleotide reduction process, evidence for its role in this process and the importance of coupling of electron and proton transfer requires new approaches.
Conclusions To further our understanding of the mechanism of di-iron cluster assembly in RNRs in vitro, an understanding of ferrous loading of R2 and the identity of the reductant in vivo is essential. The role of the Y in R2 on S formation in R1, probably involves tunneling and hopping mechanisms. Coupling of electron and proton transfer within the R1 subunit is likely given the role of hydrogen-atommediated S formation in the class II and III RNRs and the structural congruence with R1.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
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Direct detection of a W48 cation radical by EPR methods and a demonstration that its unusual spectrum is a consequence of its exchange coupling to the paramagnetic iron center. 20. Riggs-Gelasco PJ, Shu LJ, Chen SX, Burdi D, Huynh BH, Que L, Stubbe J: EXAFS characterization of the intermediate X generated during the assembly of the Escherichia coli ribonucleotide reductase R2 diferric tyrosyl radical cofactor. J Am Chem Soc 1998, 120:849-860. 21. Yun D, Krebs C, Gupta GP, Iwig DF, Huynh BH, Bollinger JM Jr: Facile electron transfer during formation of X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse. Biochemistry 2002, 41:981-990. 22. Schmidt PP, Rova U, Katterle B, Thelander L, Graslund A: Kinetic evidence that a radical transfer pathway in protein R2 of mouse ribonucleotide reductase is involved in generation of the tyrosyl free radical. J Biol Chem 1998, 273:21463-21472. 23. Bollinger JM, Krebs C, Vicol A, Chen SX, Ley BA, Edmondson DE, Huynh BH: Engineering the diiron site of Escherichia coli ribonucleotide reductase protein R2 to accumulate an intermediate similar to H-peroxo, the putative peroxodiiron(III) complex from the methane monooxygenase catalytic cycle. J Am Chem Soc 1998, 120:1094-1095. 24. Moenne-Loccoz P, Baldwin J, Ley BA, Loehr TM, Bollinger JM Jr: O2 activation by non-heme diiron proteins: identification of a symmetric mu-1,2-peroxide in a mutant of ribonucleotide reductase. Biochemistry 1998, 37:14659-14663. 25. Askwith C, Kaplan J: Iron and copper transport in yeast and its relevance to human disease. Trends Biochem Sci 1998, 23:135-138. 26. Elledge SJ, Zhou Z: Cell-cycle and DNA-damage regulation of ribonucleotide reductase in Saccharomyces cerevisiae. Induced Eff Genotoxic Agents Eukaryotic Cells 1992:65-80. 27. Zhou BBS, Elledge SJ: The DNA damage response: putting checkpoints in perspective. Nature 2000, 408:433-439. 28. Wang PJ, Chabes A, Casagrande R, Tian XC, Thelander L, Huffaker TC: Rnr4p, a novel ribonucleotide reductase small-subunit protein. Mol Cell Biol 1997, 17:6114-6121. 29. Huang MX, Elledge SJ: Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae. Mol Cell Biol 1997, 17:6105-6113. 30. Ge J, Perlstein DL, Nguyen H-H, Bar G, Griffin RG, Stubbe J: Why multiple small subunits (Y2 and Y4) for yeast ribonucleotide reductases? Toward understanding the role of Y4. Proc Natl Acad Sci USA 2001, 98:10067-10072. The first purification of all four subunits of yeast RNRs and demonstration that Y4 is essential for Y formation. 31. Nguyen H-HT, Ge J, Perlstein DL, Stubbe J: Purification of ribonucleotide reductase subunits Y1, Y2, Y3, and Y4 from yeast: Y4 plays a key role in diiron cluster assembly. Proc Natl Acad Sci USA 1999, 96:12339-12344. 32. O’Halloran TV, Culotta VC: Metallochaperones, an intracellular shuttle service for metal ions. J Biol Chem 2000, 275:25057-25060. 33. Lamb AL, Wernimont AK, Pufahl RA, Culotta VC, O’Halloran TV, Rosenzweig AC: Crystal structure of the copper chaperone for superoxide dismutase. Nat Struct Biol 1999, 6:724-729. 34. Rosenzweig AC: Copper delivery by metallochaperone proteins. Acc Chem Res 2001, 34:119-128. 35. Bar G, Bennati M, Nguyen HH, Ge J, Stubbe JA, Griffin RG: High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy: the tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast. J Am Chem Soc 2001, 123:3569-3576.
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36. Voegtli WC, Ge J, Perlstein DL, Stubbe J, Rosenzweig AC: Structure of the yeast ribonucleotide reductase Y2Y4 heterodimer. Proc Natl Acad Sci USA 2001, 98:10073-10078. The structure of the heterodimer of Y2 and Y4, active in vitro with Y1 in nucleotide reduction, is solved. No eukaryotic R2 structures have thus far been crystallized with the di-iron center in place. 37. Chabes A, Domkin V, Larsson G, Liu AM, Graslund A, Wijmenga S, Thelander L: Yeast ribonucleotide reductase has a heterodimeric iron- radical-containing subunit. Proc Natl Acad Sci USA 2000, 97:2474-2479. Purification of the four subunits of yeast RNR. The proposal is made that Y4 is a folding chaperone. 38. Thelander L, Reichard P: Reduction of ribonucleotides. Annu Rev Biochem 1979, 48:133-158. 39. Larsson KM, Andersson J, Sjoberg B-M, Nordlund P, Logan DT: Structural basis for allosteric substrate specificity regulation in anaerobic ribonucleotide reductases. Structure 2001, 9:739-750. 40. Uhlin U, Eklund H: The ten-stranded beta/alpha barrel in ribonucleotide reductase protein R1. J Mol Biol 1996, 262:358-369. 41. Sintchak MD, Arjara G, Kellogg BA, Stubbe J, Drennan CL: The crystal structure of class II ribonucleotide reductase reveals how an allosterically regulated monomer mimics a dimer. Nat Struct Biol 2001, 9:293-300. The first structure of a class II RNR. A comparison of the three-dimensional location of the B12 binding site in this protein with the G location in the class III RNR, suggests that Y730 and Y731 in R1 of the class I RNR are involved in hydrogen atom abstraction of C439 in R1 to generate a thiyl radical. 42. Stubbe J, van der Donk WA: Protein radicals in enzyme catalysis. Chem Rev 1998, 98:705-762. 43. Licht S, Gerfen GJ, Stubbe J: Thiyl Radicals in ribonucleotide reductases. Science 1996, 271:477-481. 44. Logan DT, Andersson J, Sjoberg BM, Nordlund P: A glycyl radical site in the crystal structure of a class III ribonucleotide reductase. Science 1999, 283:1499-1504. 45. Uhlin U, Eklund H: Structure of ribonucleotide reductase protein R1. Nature 1994, 370:533-539. 46. Eklund H, Eriksson M, Uhlin U, Nordlund P, Logan D: Ribonucleotide reductase – structural studies of a radical enzyme. Biol Chem 1997, 378:821-825. 47. Climent I, Sjoberg BM, Huang CY: Carboxyl-terminal peptides as probes for Escherichia coli ribonucleotide reductase subunit interaction: kinetic analysis of inhibition studies. Biochemistry 1991, 30:5164-5171. 48. Eklund H, Uhlin BE, Farnegardh M, Logan D, Nordlund P: Structure and function of the radical enzyme ribonucleotide reductase. Prog Biophys Mol Biol 2002, 77:177-268. 49. Giese B, Biland A: Recent developments of charge injection and charge transfer in DNA. Chem Commun 2002, 7:667-672. 50. Tommos C, Skalicky JJ, Pilloud DL, Wand AJ, Dutton PL: De novo proteins as models of radical enzymes. Biochemistry 1999, 38:9495-9507. 51. Ekberg M, Potsch S, Sandin E, Thunnissen M, Nordlund P, Sahlin M, Sjoberg BM: Preserved catalytic activity in an engineered ribonucleotide reductase R2 protein with a nonphysiological radical transfer pathway – the importance of hydrogen bond connections between the participating residues. J Biol Chem 1998, 273:21003-21008. 52. Ekberg M, Sahlin M, Eriksson M, Sjoberg BM: Two conserved tyrosine residues in protein R1 participate in an intermolecular electron transfer in ribonucleotide reductase. J Biol Chem 1996, 271:20655-20659.
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Di-iron-tyrosyl radical ribonucleotide reductases JoAnne Stubbe New insights have been gained into the formation of the di-iron tyrosyl radical cofactor, which is essential for nucleotide reduction in the class I ribonucleotide reductases. Recent advances include Insight into the function of the tyrosyl radical in initiation of nucleotide reduction. Address Chemistry and Biology Departments, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Current Opinion in Chemical Biology 2003, 7:183–188 This review comes from a themed section on Bioinorganic chemistry Edited by Joan Broderick and Dimitri Coucouvanis 1367-5931/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1367-5931(03)00025-5
Abbreviations RNR ribonucleotide reductase Y tyrosyl radical
Introduction Escherichia coli ribonucleotide reductase (RNR) is composed of a 1:1 complex of two homodimeric subunits: R1 and R2. R1 is the ‘business end’ of the molecule where nucleotides are converted to deoxynucleotides. R1’s active site houses the cysteines essential for catalysis and contains multiple binding sites for deoxynucleotides and ATP that govern specificity of nucleotide reduction and the rate of reduction. R2 houses the di-iron tyrosyl radical (Y) cofactor, which is essential for initiation of the nucleotide reduction process on R1. Seminal EPR experiments in conjunction with isotopic labeling methods identified this first known protein radical as a Y in E. coli R2 and established that it was essential for catalysis [1,2]. Since then, Ys or modified Ys have been shown to play essential roles in manganese-cluster-mediated O2 evolution from water in the photosynthetic reaction center II, in prostaglandin synthase, and in galactose oxidase. They also have been postulated to play a role in cytochrome oxidase and several heme-dependent peroxidases [3]. This review focuses on the di-iron-Y RNR and provides an update on our understanding of the biosynthesis in vitro and in vivo of the cofactor and its biological function.
Diferric-Y cofactor assembly in E. coli in vitro The first detailed mechanism of formation of the di-iron Y cofactor in RNR was proposed in 1991 on the basis of www.current-opinion.com
time-resolved physical biochemical methods that allowed monitoring of changes in the oxidation state of the irons during the course of the reaction (Figure 1) [4]. Highresolution structures of R2 in the diferrous form [5], the diferric form [6] and, more recently, in the presence of endogenous anions (such as azide), active site mutations (D84E [7] and double mutant Y122F/F208A [8]), and alternative metals (Mn, Co) [9,10] have played an essential role in proposing structures for intermediates along the pathway (Figure 2). These structures have demonstrated the flexibility of carboxylates E238, D84 and E204 (E. coli numbering) within the active site environment, consistent with the importance of carboxylate shifts in the chemistry of di-iron proteins [11]. Recent studies have filled in many of the missing details of the di-iron cofactor assembly mechanism. The stoichiometry of cluster assembly requires two irons and a reducing equivalent to carry out a four-electron reduction of O2 to H2O [12]. The fourth electron is supplied by tyrosine, Y122 in E. coli RNR. Recent kinetic and thermodynamic studies have suggested that one Fe binds rapidly (proposed to be Fe2, Figure 2) and tightly to apo R2 and that a slow (10 s1) conformational change limits the rate of binding of the second iron (Fe1), which binds with fivefold lower affinity [13,14]. This slow step can be bypassed by starting with R2 that has been pre-loaded with ferrous iron [15]. When the loaded diferrous R2 is mixed with O2, the first wellcharacterized intermediate detected depends on the availability of the reducing equivalent required for diiron Y formation. When the reducing equivalent is not available, an exchange-coupled intermediate composed of a tryptophan cation radical (W48Hþ) and a Fe3þ/Fe4þ di-iron cluster designated X is observed [16,17]. This W48Hþ intermediate, postulated on the basis of its absorption spectrum and the structural similarity of R2 to that in cytochrome c peroxidase [18] has recently been characterized in detail by EPR and Mo¨ssbauer methods [19]. These studies substantiated and extended the original model that either W48Hþ or X can oxidize Y122 to the Y under conditions where the reductant is limiting. Furthermore, convincing evidence was presented that the species with the absorption transient at 560 nm (putative W48Hþ) is EPR active and coupled to a second paramagnetic species X. The proposed structure for X remains unresolved, although a variety of physical methods have limited the possibilities. Recent structural studies have demonstrated that E238 is flexible and can coordinate with both Fe1 and Fe2 in a m-n1,n2 fashion [8]. While the direct relevance of these structures to the coordination of the irons during catalysis of cluster assembly is unclear, the short Fe1 to Fe2 distance (2.5 A˚) in X requires a similar coordination of at least one of the Current Opinion in Chemical Biology 2003, 7:183–188
184 Bioinorganic chemistry
Figure 1
Y122 apoR2 + Fe2+ W48
Y122 apoR2 Fe2+ W48
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Y122 Fe2+ Fe2+ W48
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?
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bridging carboxylates [20]. Thus, the crystallographic data suggest that a (m-n1,n2) E238 coordination is possible in X. Recent studies have shown the presence of intermediate X and its kinetic competence in assembly of the di-iron cofactor in mouse R2 [21], in contrast with earlier studies [22]. Thus, a congruence between eukaryotic and prokaryotic cluster assembly has been established.
O
Fe3+ W48 Native R2 3+
Fe
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Proposed mechanism for the assembly of the diferric-Y Cofactor of R2. The model is an expanded version of the original model based on recent time-resolved physical biochemical methods [14,19,23].
New insight into cluster assembly before formation of X has recently been provided. In the X-ray structure of reduced R2, both irons are four-coordinate, 3.9 A˚ apart, and E238 and E115 bridge the two irons in a m-1,3 configuration. Recent circular dichroism. magnetic circular dichroism and variable temperature, variable field magnetic circular dichroism experiments suggest, however, that Fe1 is four-coordinate and Fe2 is five-coordinate in solution, with E204 being bidentate to Fe2 [13]. A comparison of the coordination of these irons with the two irons in delta-9-desaturase and their differences in chemical reactivity with O2, suggests that a 4-coordinate/ 5-coordinate diferrous center enhances reactivity toward O2. Studies with the double mutant F208A and Y122F-R2 soaked with azide, an O2 surrogate, suggest that Fe2 is the site of O2 binding [5]. Additional experiments in which D84 is replaced with glutamic acid have provided insight into early stages of cluster assembly. Incubation of the diferrous form of the
Figure 2
(a)
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Demonstration of the flexibility of E238, D84. A comparison of (a) structures of wt R2 in the oxidized state [18] and (b) the reduced state [5] with (c) the D84E-R2 in the reduced state [7] and (d) the F208A, Y122F-R2 in the reduced state soaked with azide [8]. Current Opinion in Chemical Biology 2003, 7:183–188
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Di-iron-tyrosyl radical ribonucleotide reductases Stubbe 185
mutant with O2 generated a peroxide intermediate postulated to be on the reaction pathway in cluster assembly in wt-R2 [23]. The peroxide in the D84E-R2 has been characterized by resonance Raman [24], Mo¨ ssbauer and visible spectroscopies. Its isomer shift and visible spectrum is similar to a transient observed in wt-R2 cluster assembly [15]. Unfortunately, in the wt case, the species accounted for <10% of R2 and disappeared within 10 ms. A kinetics experiment in which the reaction is triggered with light is essential to determine if this species in wt-R2 is on the pathway and if it is structurally similar to peroxide observed with D84E-R2.
Yeast RNR: a system to examine cluster assembly in vivo Early stages of cluster assembly in R2s have been difficult to study because of inability in vitro to deliver Fe2þ and the extra reducing equivalent in a controlled fashion. Presumably, proteins play this role in vivo. Yeast is an excellent system to study cluster assembly in vitro and in vivo. Many of the genes involved in iron and copper homeostasis have been identified [25]. Genome sequences and isogenic strains of yeast with each gene deleted are available and genes for two RNRs, involved in replication and repair, have been identified [26,27]. The R1s of yeast RNR have been designated Y1 and Y3 and the R2s have been designated Y2 and Y4. Y4 is of particular interest because sequence comparison with Y2 reveals that it lacks three of the conserved iron ligands found in almost all other R2 sequences. In Y4, H118 and H241 (E. coli numbering) are each replaced with tyrosine and E238 is replaced with arginine (Figure 2) [28,29]. The arginine substitution precludes iron binding to the Fe2 site. Our hypothesis for the role of Y4 [30,31] was based on the seminal experiments in yeast, studying assembly of copper cofactors in proteins. A copper requiring protein such as superoxide dismutase has its copper delivered by a copper chaperone protein (Lys7) that is structurally homologous to superoxide dismutase [32]. Furthermore, the chaperone protein has its active site ligands modified relative to the receptor protein so that copper cannot bind. However, it also contains additional small domains that bind and deliver the copper [33,34]. The mechanism(s) for this delivery are presently under intense investigation. Our initial model proposed Y4 as a chaperone protein (Lys7 equivalent) that could deliver Fe2þ to Y2. Y4 was shown to be essential for di-iron-Y formation [30]. Incubation of Y2 and Y4 homodimers at physiological concentrations with Fe2þ and O2 in vitro resulted in rapid reorganization to generate a heterodimer Y2Y4 (one monomer of Y2 and one of Y4). Y2Y4 contained 0.6Y and 1.8 irons [35]. In vitro, the heterodimer is active with Y1 in nucleotide reduction. The structure of the heterodimer has recently been solved [36]. The structure of Y4 has also been solved to 3.1 A˚ and the structure of Y2 to 3.15 A˚ is undergoing final refinement (AC Rosenzweig et al., unpublished results). Whether Y2Y4 can re-equiliwww.current-opinion.com
brate and generate a Y2 homodimer with an active cofactor is an important unresolved issue. Studies from Thelander and co-workers [37], independently proposed that the heterodimer of Y2Y4 is active based on isolation of the Y2Y4 heterodimer when the genes for Y2 and Y4 were co-transformed in E. coli and the resulting R2 was isolated. They proposed that Y4 is a chaperone protein that prevents Y2 unfolding. Problems with both models are apparent, however, as several different yeast strains in which Y4 has been deleted are viable. While the DY4 strains are growth-compromised, their ability to grow raises the question as to the actual role of Y4 in vivo. We have recently demonstrated that one Y4 deletion strain has dramatically overproduced Y2. This strain may prove useful in purifying the factors required for cluster assembly. In E. coli, 1.2 Ys per R2 homodimer have been observed in many laboratories. In yeast, 0.6Ys per Y2Y4 heterodimer has been reported. The stoichiometry raises the possibility of half-sites reactivity of R2, which would require the R1R2 complex, essential for the nucleotide reduction process, to be unsymmetrical. In yeast, if Y2Y4 is the active species in vivo, then half-sites activity is required. Use of epitope-tagged R2s integrated into the genome under control of the wt-promoter should allow rapid removal of R2 from crude extracts and reveal if there are one or two Ys per R2. The biosynthesis of dinuclear non-heme iron systems still remains to be unraveled and the genetics and biochemistry accessible in yeast provides an excellent system to investigate this pathway. Unraveling this pathway should lead to a better understanding of the mechanism of Fe2þ binding and O2 reduction in vitro and iron deliver in vivo. Determination of the number of Ys per R2 in vivo is also essential for understanding R1R2 interactions and the details of the reduction process.
Function of Y in ribonucleotide reduction Seminal experiments in the 1970s demonstrated that Y122 of E. coli R2 is essential for the nucleotide reduction process [38]. Recently, its role in this process has been elucidated by a comparison of the structures of the three classes of RNRs [39,40,41]. RNR classifications are based on the different metallo-cofactors that initiate nucleotide reduction. The class II RNR uses adenosylcobalamin and the class III RNR utilizes a glycyl radical (G) generated by a 4Fe4S cluster and S-adenosylmethionine [42]. A comparison of the class II structure with the class I and III structures substantiated a common active site architecture where nucleotide reduction occurs. However, the structural comparison unexpectedly revealed that the location of the radical initiators in the class II and III RNRs overlapped with two conserved Ys (Y731 and Y730) in the C-terminus of R1 in the class I RNRs [41]. The mechanism of radical initiation in the Current Opinion in Chemical Biology 2003, 7:183–188
186 Bioinorganic chemistry
Figure 3
Figure 4
E441 GDP
3.4 Å C439
R2 peptide (375)
Y730
3.4 Å 3.3 Å
R2 peptide (360) R2 (340)
Y731
R2 peptide (360) R2 (340)
25 Å Current Opinion in Chemical Biology
R1 Y356?
R2 A Docking model of R1 and R2 adapted from the original model of Uhlin and Eklund [45]. In this model, R1 (a homodimer) is complementary in shape to R2 (a homodimer). In R2, the diiron center is shown as red balls. The cyan colored residues in stick format are amino acid residues proposed to play an important role in proton coupled electron transfer between R2 and R1. The C-terminus of each monomer of R2 (340) is indicated. The C-terminal 35 amino acids important in binding to R1 are disordered. Y356-R2 is a conserved residue found in this region. R1 has space-filling renditions of the substrate and effectors as indicated. The cyan-colored residues in stick format are amino acid residues proposed to play an important role in proton-coupled electron transfer between R2 and R1. The R2 peptide (360–375 in red) required for R1 to crystallize is shown.
3.1 Å 7.6 Å
The radical initiation in the class I RNRs is obviously more complex than in the other classes of RNRs. A docking model of R1 and R2 provides the working hypothesis for radical initiation: how the Y122 on R2 generates a S on R1 which is 35 A˚ distant (Figure 3) [46]. The structural model is based on the complementary shapes of R2 and R1 and the observation that the C-terminal 10–20 amino acids of R2s (disordered in all structures) are essential for binding R2 to R1 [47]. Additionally, the crystallization of R1 required the presence of the C-terminal peptide of R2, 15 residues (360–375) of which are apparent in the R1 structure [45]. The structural model, conserved residues from sequence alignments, and the long distance between Y122 on R2 and C439 on R1 suggests that the radical initiation involves a pathway with transient amino radical intermediates Current Opinion in Chemical Biology 2003, 7:183–188
Fe2 4.2 Å
Y122
class II RNR has been shown to involve hydrogen atom abstraction to generate the transient S initiator [43]. Based on the structure and chemistry, the G in the class III RNRs probably generates a putative S by hydrogen atom abstraction as well [44]. By analogy, therefore, the two Ys in the class I RNRs may be involved in hydrogen atom abstraction to generate the putative active site S [45].
D237
W48
Fe1 Current Opinion in Chemical Biology
A model for the proposed radical initiation pathway. All residues indicated are conserved. The position of Y356 relative to W48 in R2 and Y731 in R1 is unknown as it is located on the disordered C-terminus of R2 ((340) Figure 3).
(Figure 4) [48]. The rate constant for nucleotide reduction, 2–10 s1, and Marcus theory for electron transfer, require intermediates in S formation, given the long distance. Several constraints on the radical initiation process have made the mechanism difficult to study. Y is oxidized and reduced on each turnover (J Ge, personal communication) and the Y hole in R2 is the stable form of RNR. Oxidation of a cysteine by a Y is thermodynamically uphill, requiring cysteine deprotonation [42]. Cysteine oxidation is coupled to a rapid irreversible dehydration step in nucleotide reduction to facilitate the uphill process. Thus, concentrations of intermediates are expected to be low and difficult to detect. The rate-determining step in the nucleotide reduction process is a physical step gated by binding of allosteric effector and binding of substrate to R1 (J Ge, personal communication). This conformational change is www.current-opinion.com
Di-iron-tyrosyl radical ribonucleotide reductases Stubbe 187
followed by rapid electron transfer and rapid nucleotide reduction. Thus, to detect amino acid radical intermediates, the rate determining step must be changed or a trap must be placed within the pathway. The radical initiation process probably involves both tunneling and hopping mechanisms [49]. A tunneling mechanism between Y122 and W48 involving electron transfer and protonation is proposed and is based on studies of diiron Y cluster assembly on R2. As noted above, the transient W48þ can oxidize Y122 to Y122 [17,19]. The reversibility of this process hinges on the coupling of oxidation potential with protonation state of the amino acids [50] and suggests the importance of the proton transfer between W48 and D237. The inactivity of D237N-R2 [51] and the low activity of D237E-R2 support this proposal. No structural information is available about the distance between Y356 (R2) and W48 in R2 or Y731 in R1. The docking model suggests distances of 12 A˚ , well within the constraints of Marcus theory for rapid electron transfer. Mutagenesis studies have demonstrated that the conserved residues in this pathway (Figure 4) are important [51,52]. Finally, the inactivity of Y730F and Y731FR1s in the nucleotide reduction process, implicate a hydrogen atom transfer mechanism in this part of the radical initiation pathway. No amino acid radical intermediates have been detected, as required by this model. Thus, although Y122 in R2 is essential for nucleotide reduction process, evidence for its role in this process and the importance of coupling of electron and proton transfer requires new approaches.
Conclusions To further our understanding of the mechanism of di-iron cluster assembly in RNRs in vitro, an understanding of ferrous loading of R2 and the identity of the reductant in vivo is essential. The role of the Y in R2 on S formation in R1, probably involves tunneling and hopping mechanisms. Coupling of electron and proton transfer within the R1 subunit is likely given the role of hydrogen-atommediated S formation in the class II and III RNRs and the structural congruence with R1.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
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188 Bioinorganic chemistry
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