Vol. 176, No. 2, 1991 April 30, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 705-710

A MIXED VALENCE FORM OF THE IRON CLUSTER IN THE B2 PROTEIN OF RIBONUCLEOTIDE REDUCTASE FROM ESCHERICHIA COLI Michael P. Hendrich, Timothy E. Elgren and Lawrence Que, Jr. Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 Received March 14, 1991

A mixed valent form of the iron cluster (Fe(H)Fe(I/I)) in the B2 protein of ribonucleotide reductase has been isolated and characterized. The irons in this state of the protein are ferromagnetically coupled as indicated by the observation of a novel S = 9/2 EPR spectrum. This is the first ferromagnetically coupled Fe(II)Fe(III) cluster reported for a protein and the first observation of the mixed valence form of ribonucleofide reductase. ~ 1991AcademicP . . . . . I n c .

Ribonucleotide reductase (RR) catalyzes the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides, an essential step in the synthesis of DNA in all living ceils [1, 2, 3]. The Escherichia coli enzyme contains two proteins, designated B1 (~2) and B2 (132). The RRB 1 protein contains the substrate and effector binding sites as well as several redox-active thiols [4, 5, 6]. The active form of the RRB2 protein contains a stable tyrosine radical, essential for enzymatic activity, and a dinuclear nonheme iron-oxo cluster, involved in the generation and stabilization of the radical [7]. The x-ray crystal structure of the radical free, diferric B2 protein (RRB2met) was recently solved to 2.2/k resolution [8], showing two inequivalent six-coordinate Fe(III) atoms bridged by an oxo and a carboxylato group with terminal His and carboxylate ligands. The ~t-oxo bridge was previously implicated by the strong antiferromagnetic coupling (J = - 108 cm "1) of the two ferric ions [9], the large quadrupole splittings and lack of magnetic structure in M6ssbauer spectra [7], and the presence of a symmetric Fe-O-Fe vibrational mode in the resonance Raman spectrum [10]. The reported three-dimensional structure supports a stoichiometry of one dinuclear iron-oxo cluster per I] polypeptide, as previously proposed by Lynch et al. [11]. Reichard et al. have shown that aerobic incubation of RRB2met with a crude extract from E. coli will regenerate the tyrosyl radical [12]; however simple exposure to oxygen will not.

Two of the three components from this extract have been isolated and identified as superoxide dismutase [12] and an NAD(P)H:flavin oxidoreductase [13]. This observation has led to the

Abbreviations used here: RR, ribonucleotide reductase; Hr, hemerythrin; EPR, electron paramagnetic resonance; EDTA, ethylenediaminetetraacetate; HEPES, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid. 0006-291X/91 $1.50 705

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proposed mechanism that the oxidoreductase may be capable of reducing RRB2 to a form which could then react with dioxygen followed by oxidation of Tyr122. The tyrosyl radical may also be generated by exposure of the diferrous (RRB2red) form of the protein to dioxygen [9] or the aerobic Fe(II) reconstitution of metal-free (RRB2apo) protein [7, 14]. Mixed valence (Fe(l/)Fe(llI)) forms of dinuclear nonheme iron clusters have been identified and spectroscopically characterized for such metalloproteins as hemerythrin [15], the hydroxylase component of methane monooxygenase [16] and the purple acid phosphatases [17]. The mixed valence form of the iron clusters in RRB2 has been proposed [18, 19] as a reactive intermediate generated during the activation of dioxygen catalyzed by RRB2red; however it has not been previously observed despite attempts to do so via chemical reduction of RRB2met [18]. Therefore, we have pursued a novel approach in the stabilization of mixed valent species. This approach has provided the first direct spectroscopic evidence for a mixed valent state of the iron cluster in RRB2 (RRB2mv). MATERIALS AND METHODS RRB2 was isolated from E. coli strain N6405/pSPS2 [20], a heat-inducible overproducer, as previously described [21] and concentration determined by absorbance at 280 nm (E280= 141 mM-lcm -1) [11]. The RRB2 protein was purified to homogeneity as indicated by the presence of a single band on an SDS-PAGE electrophoresis gel. Hemerythrin was isolated from Phascolopsis gouldii as previously described [22]. Methemerythrin was prepared by oxidation of deoxyhemerythrin with K3Fe(CN)6; the concentration was determined spectrophotometrically (E333 = 65 mM-lcm -1) [23]. All solutions were prepared with 25 mM HEPES buffered at pH 7.6. EPR spectra were collected on a Varian El09 spectrometer equipped with an Oxford ESR10 cryostat for low temperature studies. The double integrated EPR signals were quantified relative to a Cu(II)EDTA standard. X-irradiation was performed with a GE x-ray tube (Mo target) operating at 30 kV and 20 mA. Total irradiation time was typically 4 hours. RESULTS AND DISCUSSION Samples of the diferric form of RRB2 (RRB2met) were exposed to ionizing radiation from an x-ray source while immersed in liquid nitrogen. An EPR spectrum at T = 3 K of RRB2met after irradiation is shown in Figure la. The intense resonances in the range 2.2 > g > 1.8 (g = hv/13B) are due to peptide-based free radicals and solvated electrons generated by the irradiation [24]. As the temperature of an irradiated sample is successively raised to higher temperatures, Tanneal, and then lowered to T = 3 K for spectral measurement, no changes are observed in the 3 K EPR signals for Tanneal < 150 K. However, for Tanneal = 200 K, new signals are observed with features at g = 14.0, 6.6, and 5.4. Figure lb shows a difference spectrum obtained from the subtraction of the Tanneal = 200 K spectrum minus the spectrum obtained before annealing. The inversion of intensity of the g = 2 resonances indicates that the radical species giving rise to these signals have either lost or recovered an electron during the annealing process. Some of these radical signals are off the scale of Figure 1 and thus the signals have been deleted for clarity. The low field signals persist until the sample is thawed, after which the EPR spectrum of the sample before irradiation is recovered (not shown). The same sample when cycled through the irradiation/anneal process again gives the signals of Figure 706

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( 1.94

.... _JF_ 5.4

14.0

I

en

?

6.6

I

(1.87

) 1.96

I 1.67

i

0

100

'

200

'

3~)0

'

400

'

500

B(mT)

Figure 1, X band EPR spectra of mixed valent ribonucleotide reductase [1.9 mM] and semi-met hemerythrin [2.2 mM] at T = 3 K generated with x-irradiation and annealing. (a) Difference spectrum (after-before irradiation) of RRB2met. (b) Difference spectrum (after-before anneal at 200 K) of the irradiated RRB2met sample of (a). (c) Difference spectrum of (semi-met)Rt-Ir. The dashed spectrum in (b) is a simulation for the EPR spectrum of the ground Kramers doublet of a S = 9/2 multiplet. The calculation uses a S = 9/2 spin Hamiltonian with D > 0, E/D ---0.03, and 6E/D = 0.015 [Hs = D(Sz2 - 33/4) + E(Sx2 - Sy2) + I3B.g.S]. We assume the EPR linewidth is dominated by a spread (Gaussian) in the rhombicity E/D with width OE/D(one standard deviation). All spectra in the close vicinity of g = 2 have intense resonances from radical species; these signals are off scale and have been deleted for clarity. EPR instrumental parameters: microwaves, 9.142 GHz at 0.20 mW; modulation, 100 kHz at 10 Gpp; gain, lxl04 (a,b) or 2.5x104 (c).

lb. The low field signals showed no differences between aerobically and anaerobically prepared protein samples. The low field signals of Figure l b originate from a novel mixed valent iron species of RRB2mv. W e propose this species is trapped at temperatures Tanneal = 200 K when an electron migrates to the diferric site o f RRB2met resulting in a one electron reduced iron cluster. These low field signals are indicative of a specific spin state of an exchange coupled, mixed valent diiron center. For a spin S = 9/2 center with a positive zero-field parameter D, and rhombicity E/D = 0.03, an effective g-value for the Kramers doublet having lowest energy occurs at g = 14 and for the first excited doublet near g = 5.4. A temperature study of the low field signals of Figure l b indicate that indeed the g = 14 resonance is from the ground doublet and the g = 5.4 resonance from an excited doublet. A fit to the temperature data indicates a value for D(S = 9/2) in the range 2 < D < 6 cm -1. The S = 9/2 assignment is confirmed by the simulation of the ground doublet spectrum which is superimposed on Figure lb. The spectral calculation uses an effective S'= 1/2 doublet representation, with g-tensor and g-strain tensor based on a S = 9/2 spin Hamiltonian having just two adjustable parameters: E/D and C~E/D(see Figure 1). The simulation facilitates quantitation of a spectrum which overlaps with many other resonances. 707

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A spin S = 9/2 center for RRB2 can originate from a ferromagnetic exchange coupling between a spin S1 = 5/2 site (high-spin ferric) and a spin $2 = 2 site (high-spin ferrous) [25, 26] 1 Quantitation versus a copper standard indicates 0.5% of the RRB2 diiron centers account for the mixed valent EPR signal. The low fraction of mixed valent centers generated by this technique is expected, because most of the electrons generated by the x-irradiation presumably recombine with the nearest electron hole. To test that the 0.5% mixed valent species is not an x-ray generated artifact, we have performed the same experirnents on a sample of methemerythrin (metHr). No EPR signal changes are observed for Tanneal < 170 K. For Tanneal = 200 K, the signal characteristic of (semimet)RHr (one electron reduced metHr) is observed [15]; the difference spectrum is shown in Figure lc. Quantitation of the (semimet)RHr signal indicates that 1% of the diiron centers account for the mixed valent signal. The generation of (semimet)RHr via irradiation is an important control experiment from which we draw three conclusions. First, the relative amount of mixed valent iron center generated from RRB2met or from metHr is of the same order of magnitude. Therefore, the low yield of the mixed valence species for both RRB2 and Hr is a result of the relative inefficiency of this technique under these experimental conditions. Second, the S = 1/2 (semimet)RHr EPR signal shown here, which originates from an antiferromagnetically coupled Fe0I)Fe(III) center, is the same as that generated by standard chemical reduction methods. Therefore, the ferromagnetic coupling observed for RRB2mv is a result of the particular nature of the RRB2 iron pair and its unique protein environment. This conclusion is of particular importance since all the IX-OXOiron proteins and chemical models studied to date have diiron centers which are antiferromagnetically coupled when the mixed valent state is observed [27]. Third, it is possible that the S = 9/2 center could be the result of a ferromagnetically coupled, Fe(III)Fe(IV) species, if an electron is abstracted from the diiron center by a radical species that is close to an iron atom. However, this is clearly not the case for the Hr study; therefore we suspect the mixed valent RRB2 species is Fe(II)Fe(III). The feature at g = 6.6 in Figure lb is from an excited doublet, but its resonance position is not predicted by the simplistic assumptions of a S = 9/2 center. 2Further studies are under way to understand the origin of the g = 6.6 resonance, to significantly increase the fraction of centers giving the mixed valent signal, and to identify the molecular species which donates the electron to the iron center. In particular, the resonance at g = 1.94 in Figure la is correlated to the mixed valent species. The S = 9/2 signals are observed to grow in over several minutes at

1 Spin S = 9/2 centers have been observed from a bis I.t-phenoxo Fe(II)Fe(III) complex [25] and a tri ~t-hydroxo Fe(II)Fe(III) complex [26]. For the latter complex the valence is delocalized (Fe2.5+Fe2.5+), in which case a double exchange interaction must be included with the standard Heisenberg exchange interaction to interpret the magnetic data. Further studies are needed to determine whether the RRB2mv signal is from a trapped or delocalized valence state of the iron pair. 2 For a sufficiently small exchange interaction, the S = 9/2 assumption must be replaced by a more complicated model, which may possibly explain the g = 6.6 feature. Alternatively, the magnetic anisotropy of the ferrous site may result in a shift of a principal g-value. 708

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Tanneal = 200 K. Over this same time period the g = 1.94 radical signal decays in proportion to the growth of the S = 9/2 signals. The mixed valent form of RRB2 is suspected as a possible short-lived intermediate in the generation of the tyrosine radical [18, 19]. These studies have allowed the characterization of such an intermediate and should promote the identification of this species by chemical and other spectroscopic means. RRB2 is the first kt-oxo diiron protein for which the diiron center is ferromagnetically coupled in the mixed valent state. Two model complexes are currently available which afford a S = 9/2, mixed valent center [25, 26]. The oxidized form of these complexes differ significantly from RRB2met, not only in bridging species but also in the number of terminally coordinated nitrogen and oxygen ligands. Presumably, the kt-oxo bridge is modified in RRB2mv, resulting in an altered exchange coupling. However it is unclear whether this alone is sufficient to explain the large change of more that 100 cm -I in the coupling upon one electron reduction. ACKNOWLEDGMENTS This work was supported by grants from the National Institute of Health, GM-12996 (M. P. H.), and from the National Science Foundation, DMB-8314935, DMB-8804458 (L. Q.). We thank Professor Joanne Stubbe for providing E. coli strain N6405/pSPS2 used in these experiments. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Reichard, P. and Ehrenberg, A. (1983) Science 221, 514-519. Stubbe, J. (1990) J. Biol. Chem. 265, 5329-5332. Sj6berg, B.-M. and Gr~islund, A. (1983) Adv. Inorg. Biochem. 5, 87-110. von D6beln, U. and Reichard, P. (1976) J. Biol. Chem. 251, 3616-3622. Brown, N. C. and Reichard, P. (1969) J. Mol. Biol. 46, 39-55. Thelander, L. (1974) J. Biol. Chem. 249, 4858-4862. Atkin, C.; Thelander, L.; Reichard, P. and Lang, G. (1973) J. Biol. Chem. 248, 74647472. Nordlund, P.; Sj6berg, B.-M. and Eklund, H. (1990) Nature 345, 593-598. Petersson, L.; Graslund, A.; Ehrenberg, A.; SjOberg, B.-M. and Reichard, P. (1980) J. Biol. Chem. 255, 6706-6712. Sj/3berg, B.-M.; Loehr, T. M. and Loehr, J. S. (1982) Biochemistry 21, 96-102. Lynch, J. B.; Juarez-Garcia, C.; Mfinck, E. and Que, L., Jr. (1989) J. Biol. Chem. 264, 8091-8096. Eliasson, R.; J6rnvall, H. and Reichard, P. (1986) Proc. Natl. Acad. Sci. USA 83, 23732377. Fontecave, M.; Eliasson, R. and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331. Brown, N. C.; Eliasson, R.; Reichard, P. and Thelander, L. (1969) Eur. J. Biochem. 9, 512-518. Babcock, L. M.; Bradic, Z.; I-Iarrington, P. C.; Wilkins, R. G. and Yoneda, G. S. (1980) J. Amer. Chem. Soc. 102, 2849-2850. Fox, B. G.; Surerus, K. K.; Mfinck, E. and Lipscomb, J. D. (1988) J. Biol. Chem. 263, 10553-10556. Antanatis, B. C. and Aisen, P. (1982) J. Biol. Chem. 257, 5330-5332. Sahlin, M.; Gr/islund, A.; Petersson, L.; Ehrenberg, A. and Sj6berg, B.-M. (1989) Biochemistry 28, 2618-2625. Elgren, T. E.; Lynch, J. B.; Juarez-Garcia, C.; MiJnck, E.; SjOberg, B.-M. and Que, L., Jr. (1991) J. Biol. Chem., submitted for publication. 709

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20. Salowe, S. P. and Stubbe, J. (1986) J. Bacteriol. 165, 363-366. 21. Salowe, S. P. (1987) "Chemistry of the binuclear iron/tyrosyl radical cofactor of ribonucleotide reductase from E. coli" Ph.D. thesis, University of Wisconsin, Madison. 22. Maroney, M. J.; Kurtz, D. M., Jr.; Nocek, J. M.; Pearce, L. L.; Que, L., Jr. (1986) J. Amer. Chem. Soc. 108, 6871-6879. 23. Garbett, K.; Damall, D. W.; Klotz, I. M. and Williams, R. J. P. (1969) Arch. Biochem. Biophys. 135, 419-434. 24. Box, H. (1977) Radiation Effects: ESR andENDOR Analysis; Academic Press; NewYork. 25. Surerus, K. K.; Munck, E.; Snyder, B. S. and Holm, R. H. (1989) J. Amer. Chem. Soc. 111, 5501. 26. Ding, X.; Bominaar, E. L.; Bill, E.; Winkler, H.; Trautwein, A. X.; Drueke, S.; Chaudhuri, P. and Wieghardt, K. (1990) J. Chem. Phys. 92, 178-186. 27. Que, L., Jr. and True, A. E. (1990) Prog. Inorg. Chem. (Lippard, S., Ed.) 38, 97-200.

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