Proc. Natl. Acad. Sci. USA Vol. 96, pp. 8979–8984, August 1999 Biochemistry

High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5؅-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase

CHRISTOPHER C. LAWRENCE*, MARINA BENNATI†,HONORIO V. OBIAS*, GALIT BAR†,ROBERT G. GRIFFIN†‡, AND JOANNE STUBBE*‡

*Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and †Department of Chemistry and Massachusetts Institute of Technology/Harvard Center for Magnetic Resonance, Francis Bitter Magnet Laboratory, Cambridge, MA 02139

Contributed by JoAnne Stubbe, June 4, 1999

ABSTRACT Class I ribonucleotide reductases (RNRs) nucleotide analogs provided direct evidence that the thiyl are composed of two subunits, R1 and R2. The R2 subunit radical generates a 3Ј-nucleotide analog radical (13–16). Once contains the essential diferric cluster–tyrosyl radical (Y⅐) 1 is generated, there is also excellent model precedent that a cofactor and R1 is the site of the conversion of nucleoside ketyl radical 2 is generated subsequent to loss of water from the .(diphosphates to 2؅-deoxynucleoside diphosphates. A mutant 2Ј position of the nucleotide (17, 18 in the R1 subunit of Escherichia coli RNR, E441Q, was The mechanism for the reduction of 2 has received less generated in an effort to define the function of E441 in the experimental attention. Biochemical and crystallographic ex- nucleotide-reduction process. Cytidine 5؅-diphosphate was periments indicate that C225 and C462 in the Escherichia coli incubated with E441Q RNR, and the reaction was monitored class I RNR are oxidized concomitant with nucleotide reduc- by using stopped-flow UV-vis spectroscopy and high- tion (4, 5, 19, 20). We have proposed that this reaction frequency (140 GHz) time-domain EPR spectroscopy. These proceeds by an electron-coupled proton transfer to 2 from the studies revealed loss of the Y⅐ and formation of a disulfide thiolate of C225 and the sulfhydryl of C462 to form a 3Ј- radical anion and present experimental mechanistic insight ketodeoxynucleotide 3 and a three-centered bond, the disul- into the reductive half-reaction catalyzed by RNR. These fide radical anion (8, 21). Compound 3 is also proposed to be results support the proposal that the protonated E441 is reduced by electron-coupled proton transfer. The resulting required for reduction of a 3؅-ketodeoxynucleotide by a disul- 3Ј-deoxynucleotide radical 4 would then be rapidly reduced to fide radical anion. On the minute time scale, a second radical generate product 5 and to regenerate the thiyl radical on C439. species was also detected by high-frequency EPR. Its g values The importance of coupling the proton transfer to the electron suggest that this species may be a 4؅-ketyl radical and is not transfer thermodynamically in step 4 (Scheme 1) of the on the normal reduction pathway. These experiments demon- reduction process has recently been pointed out (10, 17), and strate that high-field time-domain EPR spectroscopy is a the structure of class I RNR suggested that the protonated powerful new tool for deconvolution of a mixture of radical glutamate plays an important role in this process (5, 17). species. The potential role of the glutamate (acid) in both the chemistry of water loss and ketone reduction provided the Ribonucleotide reductases (RNRs) catalyze the conversion of impetus to make and investigate a glutamate 3 glutamine nucleotides to deoxynucleotides in all organisms (1–3). These mutation of residue 441 (22). The same mutant was also enzymes have been divided into four classes based on the recently prepared by Sjo¨berg and coworkers (23). We have metallo-cofactor required to initiate the radical-dependent predicted that the slow chemical step in the conversion of nucleotide-reduction process. Recent structural studies on NDPs to dNDPs is the reduction of 3 by the disulfide radical class I and class III RNRs (4–6) support the original proposal anion (10). Mutation of the glutamate to glutamine would thus of Stubbe and coworkers that, despite the differences in the be expected to allow buildup of this intermediate in sufficient metallo-cofactors in the various classes of RNRs, the function quantities that it might be detectable. In this paper, we report of each cofactor is to generate a thiyl radical that, via a direct spectroscopic evidence for the disulfide radical anion in common mechanism, initiates nucleotide reduction by 3Ј- the class I RNRs. hydrogen atom abstraction (Scheme 1) (7–9). A further com- monality between class I and II RNRs is the presence of a MATERIALS AND METHODS conserved glutamate residue adjacent to the two cysteine residues that deliver the reducing equivalents essential for the EPR tubes for 9-GHz spectroscopy (quartz, o.d. 4 mm, i.d. 2.4 reduction process (5). This paper provides evidence that the mm) and for 140-GHz spectroscopy (silica, o.d. 0.55 mm, i.d. role of this glutamate (in the protonated form) is to facilitate 0.40 mm) were purchased from Wilmad (Buena, NY). The the reduction of the 3Ј-ketodeoxynucleotide 3 (Scheme 1) by mutant E441Q R1 (inactive with respect to nucleotide reduc- a disulfide radical anion intermediate. tion) and R2 (specific activity 5,800–8,000 nmol⅐minϪ1⅐mgϪ1) Much is known about the initial stages of the reduction were grown and purified as described (22, 24). Their concen- ␧ ϭ process (1, 10). There is direct evidence that in the class II, trations were measured spectrophotometrically by using 280 adenosylcobalamin-dependent RNRs, the metallo-cofactor 189,000 MϪ1⅐cmϪ1 and 130,500 MϪ1⅐cmϪ1 for R1 and R2, generates a thiyl radical in a kinetically-competent fashion (11, respectively. The Y⅐ content of R2 was assessed by the drop- 12). Recent studies with a class I RNR containing a diferric ⅐ tyrosyl radical (Y ) cofactor and a class II RNR using cytidine Abbreviations: RNR, ribonucleotide reductase; SF, stopped-flow; Y⅐, the tyrosyl radical on the R2 subunit of E. coli RNR; CDP, cytidine Ј The publication costs of this article were defrayed in part by page charge 5 -diphosphate. ‡To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked ‘‘advertisement’’ in Chemistry, Massachusetts Institute of Technology, 77 Massachusetts accordance with 18 U.S.C. §1734 solely to indicate this fact. Avenue, Cambridge, MA 02139. E-mail: [email protected] or PNAS is available online at www.pnas.org. [email protected].

8979 Downloaded by guest on October 2, 2021 8980 Biochemistry: Lawrence et al. Proc. Natl. Acad. Sci. USA 96 (1999)

Scheme 1.

line correction method (25). E441Q R1 was prereduced as the final concentration of each component was the same as described (20). indicated above, and each syringe contained 15 mM Mg(OAc)2 Stopped-flow (SF) studies were carried out at 25°C by using and 50 mM Tris (pH 7.6). an Applied Photophysics (Surrey, U.K.) DX.18MV SF spec- EPR Spectroscopy. For preparation of samples to be ana- trophotometer. Data was collected in either diode-array mode lyzed at 9 GHz, a 150-␮l solution of 150 ␮M prereduced E441Q or in single-wavelength mode. In the latter case, a minimum of R1, 100 ␮MR2(inY⅐), 0.2 mM dTTP, 5 mM DTT, and 15 mM three traces were used to obtain an average for each condition Mg(OAc)2 in 50 mM Tris (pH 7.6) was placed in an EPR tube. tested. Linear and nonlinear least-squares fits to SF data were An equal volume of 3.34 mM CDP, 0.2 mM dTTP, 5 mM DTT, carried out by using either the Applied Photophysics system and 15 mM Mg(OAc)2 in 50 mM Tris (pH 7.6) was then added. software or KALEIDAGRAPH. Diode-array SF spectra were After mixing, the solution was frozen at t ϭ 10sort ϭ 3 min analyzed with SPECFIT (Spectrum Software Associates, Chapel by immersion into a bath of liquid isopentane cooled to HIll, NC) or PRO-K (Applied Photophysics) software. Ϫ140°C by a surrounding jacket of liquid nitrogen. Several EPR spectra at 9 GHz were acquired on a Bruker ESP-300 control experiments were carried out. In one control, a solu- spectrometer at 100 K. EPR spectra at 140 GHz were acquired tion of 200 ␮M prereduced E441Q R1, 3.34 mM CDP, 0.2 mM with a custom-designed high-sensitivity pulsed spectrometer dTTP, and 15 mM Mg(OAc)2 was prepared in 50 mM Tris (pH (26). Typical ␲/2 pulses of 70 ns were achieved by using a 7.6), and the reaction commenced by addition of an equal high-quality (Q Ϸ 3,000) cylindrical cavity with an incident volume of 200 ␮MR2(inY⅐), 0.2 mM dTTP, and 15 mM microwave power of 5 mW. The absolute spin sensitivity at low Mg(OAc)2 in 50 mM Tris (pH 7.6). A second control revealed temperatures has been previously determined to be 3 ϫ 109 that the trapped intermediates were stable for at least 3 weeks spins⅐GϪ1 (26) and allows for detection of ␮M spin concen- when stored in liquid nitrogen. Samples to be analyzed by EPR trations in less than a microliter volume. Pulsed EPR spectra spectroscopy at 140 GHz were prepared in a fashion similar to were acquired by using a standard three-pulse stimulated echo that described above except that the final volume was 10 ␮l and ϭ ␶ ϭ ␶ ϭ sequence (t␲/2 72 ns, pulse spacing 1 2 230 ns) and the final concentrations of R1, R2 (in Y⅐), and dTTP were 282 integrating the echo intensity over the magnetic field sweep. ␮M, 282 ␮M, and 0.8 mM, respectively. Samples were drawn Experimental conditions, such as the number of transients per into a sample tube by capillary action to a height of Ն2 mm, point, number of scans, and recycle delays were adjusted for frozen in isopentane, and stored in liquid nitrogen. The small different signal intensities and temperature and are reported sample volume required special precautions to prevent warm- in the figure legends. ing while loading the sample into the cavity. The EPR probe SF UV-Vis Spectroscopy. Syringe A contained 75 ␮M was cooled by immersion in liquid nitrogen, and the sample Ј prereduced E441Q R1, 3.34 mM cytidine 5 -diphosphate tube was loaded into the TE011 cylindrical resonator of the (CDP), 0.2 mM dTTP, 10 mM DTT, and 15 mM Mg(OAc)2 EPR probe under liquid nitrogen before being placed inside in 50 mM Tris (pH 7.6). Syringe B contained 75 ␮M R2 (based the Dewar flask, cooled to 80 K. ⅐ on Y ), 0.2 mM dTTP and 15 mM Mg(OAc)2 in 50 mM Tris Spin quantitation at 9 GHz was achieved by double inte- (pH 7.6). The reaction was commenced by rapidly mixing 25 gration and comparison to a 100 ␮M sample of R2 whose Y⅐ ␮l from each syringe and was monitored at 412 nm for 20 s or content had been established by using UV-vis spectroscopy. 100 s by using a split time base (500 points from 0 to 0.5 s, 500 Spectra of quenched samples and of the Y⅐ standard were points from 0.5–20 s or 100 s). The experiment was also recorded under identical conditions as given in the text. Data performed in diode-array mode by using the same syringe manipulations were performed by using KALEIDAGRAPH soft- contents as above and recording 800 spectra from 350 to 700 ware. nm for 30 s. Three alternative mixing modes were also inves- tigated. In case one, prereduced E441Q R1, CDP, and dTTP RESULTS in one syringe was mixed with R2 and dTTP in a second syringe. In case two, E441Q R1, dTTP, and DTT was mixed Radical Species Generated During the Incubation of E441Q with R2, CDP, and dTTP. In case three, E441Q R1, R2, dTTP, R1 RNR with CDP and dTTP. Recent studies by Persson et al. and DTT was mixed with CDP, dTTP, and DTT. In each case, (27) reported that incubation of E441Q R1 RNR with CDP Downloaded by guest on October 2, 2021 Biochemistry: Lawrence et al. Proc. Natl. Acad. Sci. USA 96 (1999) 8981

and the allosteric effector dTTP resulted in loss of the Y⅐ straightforward, because this species does not change during accompanied by formation of at least two new radical species. the course of the experiment. Subtraction of the Y⅐ is more These species were examined by 9-GHz EPR spectroscopy. complex because the amount lost during the 10-s incubation is The initial radical they observed, at 10 s, was a composite of not easy to quantify because the Y⅐ and the new radical have Y⅐ and at least one new radical. On subtraction of the spectral overlap in both the visible spectrum and in the EPR predominant Y⅐, the spectrum of the new radical was reported spectrum (see below). The results of a spectroscopic subtrac- to have a gav of 2.01. These workers concluded that this new tion in which the initial visible spectrum (obtained at 2.5 ms) radical species was the C439 thiyl radical generated concom- is subtracted from that obtained at 10 s (Fig. 2) reveals a itant with loss of the Y⅐ (27). The reported g values, however, spectrum similar to those previously reported for disulfide are inconsistent with their thiyl radical assignment (28–31). In radical anions (32, 33). an effort to further support their thiyl radical assignment, EPR Spectrum at 9 and 140 GHz of the 10-S Radical. As Persson et al. repeated this experiment with ␤-2H-labeled described above, with 9 GHz EPR spectroscopy, Persson et al. cysteine E441Q R1 (27). The perturbation of the EPR spec- (27) detected a new radical with a gav of 2.01 at 10 s. However, trum confirmed that this radical is in fact cysteine-based and the superposition of three powder spectra—the new signal provided the impetus for us to repeat their experiments. observed at 10 s, the predominant Y⅐, and an additional signal To determine the most appropriate time to quench the to be discussed below—made assembly of the unique spectrum reaction for examination by EPR methods, we performed a SF of the 10-s radical challenging given the spectral overlap. Using ␭ experiment monitoring the reaction at 412 nm, the max of the the same conditions and using similar subtraction methods, we Y⅐. The results of a typical experiment are shown in Fig. 1. The reproduced their results. A control experiment with prere- kinetic changes are complex. In contrast to expectations, A412 duced E441Q R1 in place of E441Q R1 and DTT ensured that increases and then decreases in a much slower phase. The this new feature was not associated with the DTT in the maximum absorbance is reached at Ϸ5 s, and a fit of the data reaction mixture. Analysis of the amount of total radical between0and5stoasingle exponential gives a rate constant present in the sample quenched at 10 s reveals that 20 Ϯ 5% Ϫ1 of 0.8 s . The decrease in A412 from 10 to 100 s can also be ⅐ Ϫ of the starting Y has been lost but that it is not quantitatively fitted to a single exponential with a rate constant of 0.035 s 1. converted into the new radical species. Once again, this A 9-GHz EPR analysis of a sample quenched at 10 s reveals quantitation is complicated by the presence of spectral overlap loss of 20 Ϯ 5% of the initial Y⅐ in addition to formation of the of multiple species. radical species described by Persson et al. (27). The observa- To establish accurate g values for this new species, we ⅐ ␧ ϭ tion of an increased A412 despite loss of the Y ( 1,800 recorded the spectrum at 140 GHz, where the 15-fold higher Ϫ1⅐ Ϫ1 M cm ) suggests that the new radical species must have an g factor resolution (relative to 9 GHz) permits us to distinguish ⅐ A412 with a larger extinction coefficient than the Y . The between the radical of interest and the remaining Y⅐ (Fig. 3A). kinetics and amounts of the intermediate were unaffected by On the low-field side, two g values of 2.023 and 2.015 of the new changing the order of mixing of the reagents or by using radical are readily apparent. At high field, the Y⅐ (Fig. 3B) prereduced E441Q R1 in the absence of an external reductant obscures the third g value, and its high intensity relative to the (DTT). new radical once again makes difference spectroscopy prob- To examine these changes more carefully and to determine lematic. the visible spectroscopic characteristics of this (these) new Separation of EPR Signals by Time-Domain EPR Spectros- species, we carried out a similar SF experiment with detection copy. To obtain the EPR signals of the new radical(s) gener- by using a diode-array spectrometer (data not shown). A new ated during incubation of E441Q R1 with R2 and CDP, it is species clearly formed and decayed in the region between 350 necessary to filter the intense signal of the remaining Y⅐. This and 500 nm. Examination of the proposed mechanism of is possible with time-domain techniques that exploit the en- ⅐ nucleotide reduction (Scheme 1) reveals two species, in addi- hanced spin–lattice relaxation time (T1)oftheY located on tion to the Y⅐ on R2, that have absorption features in the visible ␭ ϭ ␧ ϭ region: a thiyl radical ( max 325–330 nm ( 300–600 Ϫ1⅐ Ϫ1 ␭ ϭ M cm ) and a disulfide radical anion ( max 400–450 nm (␧ ϭ 8,000–15,000 MϪ1⅐cmϪ1) (32). To obtain the visible spectrum of this new species, one needs to subtract contribu- tions to the spectrum from the diferric cluster and the Y⅐ of R2. Both of these species have been well characterized (25). Subtraction of the contribution from the diferric cluster is

FIG. 2. Diode-array SF UV-vis spectroscopy: Difference spectrum of the 10-s intermediate. Detection was made by diode array, and data (800 scans) were acquired to 32 s (2.56-ms integration time) on a linear time scale. The first spectrum was recorded 2.5 ms after mixing (instrument dead time). The difference spectrum (solid line) was obtained by subtraction of the starting spectrum from the spectrum recorded at 10 s. To obtain the complete spectrum of the intermediate, a proportion of the Y⅐ (dotted line) must be added to the difference spectrum, because EPR analysis reveals loss of Y⅐ during the first 10 s. FIG. 1. SF UV-vis spectroscopy at 412 nm. E441Q R1 (75 ␮M), The dashed line shows the spectrum obtained by adding 0.23 equiv- CDP (3.34 mM), dTTP (0.2 mM), and DTT (10 mM) in Tris buffer alent of Y⅐ to the difference spectrum. The exact amount of Y⅐ lost were mixed with an equal volume of R2 (75 ␮M) in the same buffer. during the first 10 s is difficult to quantitate because of spectral overlap Data (1,000 points) were acquired to 20 s. in both the EPR and UV-vis spectra. Downloaded by guest on October 2, 2021 8982 Biochemistry: Lawrence et al. Proc. Natl. Acad. Sci. USA 96 (1999)

decay after the first microwave pulse. The EPR signals of other radicals with longer relaxation times, however, remain visible. Thus, this T1 filter permits suppression of the spectrum of the predominant Y⅐ (90%) and observation of the EPR spectra of the radicals of interest. Fig. 3C shows the echo-detected 140-GHz EPR spectrum at 60 K of the reaction quenched 10 s after mixing. The low-field side of the spectrum remains unchanged relative to that obtained at 20 K (Fig. 3A). However, on the high-field side, the characteristic features of the Y⅐ have disappeared, and a new axially asymmetric powder pattern is visible. A comparison with spectra of samples of the reaction mixture quenched at 3 min after mixing reveals that, in addition to the sulfur-centered radical, the 10-s sample also contains significant amounts of a second radical observed at later times (Fig. 3D). Subtraction of this second species from the spectrum in Fig. 3C reveals the complete spectrum of the sulfur-centered radical (Fig. 3E). The powder pattern is dominated by an axially asymmetric g ϭ ϭ anisotropy with principal values of g1 2.023, g2 2.015, and ϭ g3 2.002. These g values eliminate a thiyl radical (28–30) and a persulfide radical (28, 36, 37) as the source of this signal and are consistent with a disulfide radical anion (Table 1) (28, 38–40). Finally, we also note in the low-field edge of the spectrum a long, weak tail (Fig. 3E). The origin of this intensity is unknown but could possibly arise from the disulfide radical anion in equilibrium with a thiyl radical (Scheme 2) (41–43). A simulated rigid-limit powder pattern of the putative disulfide radical anion intermediate, including two isotropic hyperfine constants of 10 G, is shown in Fig. 3F. We believe that the deviations in the relative intensities at g1 and g3 may arise from anisotropic spin–spin relaxation, which typically affects the FIG. 3. Stimulated echo-detected EPR spectra at 140 GHz of the line shape at higher temperatures. E441Q R1 reaction quenched 10 s after mixing. (A) Single-scan A Second EPR Active Species Observed at 3 Minutes. The spectrum at 20 K collecting 300 transients per point. The pulse report of Persson et al. (27) described a second EPR active spacings are ␶1 ϭ ␶2 ϭ 230 nsec, and the repetition rate is 10 ms. The species kinetically linked to their putative thiyl radical. The fact Inset shows an average of 20 scans, magnified 20-fold. (B), The that the species was nucleotide-based was confirmed in an spectrum of the R2 Y⅐ recorded under the same conditions as in A.(C) experiment by using [U-13C]CDP. They proposed that this new Electron-spin echo-detected spectrum of the same mixture as A at 60 species was the 3Ј-nucleotide radical 1. Given that the first K collecting 300 transients per point; the low-field (49,050–49,570 G) radical is not the C439 thiyl radical, it is unlikely that the and high-field (49,570–49,900G) parts of the spectrum were recorded with steps of 2 G and 1 G, respectively. (D) Reference spectrum of the second radical could be 1. We therefore recorded a high-field radical present after 3 min of incubation (see Fig. 4B). (E) Spectrum (140 GHz) pulsed-EPR spectrum of this second radical as of the putative disulfide radical anion obtained by subtraction of shown in Fig. 4B. Once again, the three-pulse echo filter spectrum D from spectrum C.(F) Calculated powder pattern for g1 ϭ permits the spectrum of the nucleotide-based radical of inter- ϭ ϭ 2.023, g2 2.015, and g3 2.002 and two isotropic hyperfine couplings est to be obtained devoid of the remaining Y⅐. The spectrum ϭ ϭ a1 a2 10 G. The stimulated echo pulse sequence used is shown at is identical to the one obtained by more conventional differ- the top of the Figure. ence spectroscopy (Fig. 4A). The g values of the nucleotide- ⅐ based radical are estimated from the main turning points of the R2. Analysis of the relaxation behavior of the Y has previously derivative spectrum and are 2.0072, 2.0061, and 2.0021, giving indicated that the short T1 is due to magnetic dipolar and a gav of 2.0051. From published data (18, 44–46) discussed exchange interactions with the adjacent diferric cluster (34). below, it is clear that this new radical cannot be a 3Ј nucleotide Furthermore, in 140-GHz CW experiments, we have previ- radical as originally proposed (27). The g values suggest that it ⅐ ously noticed that the Y does not saturate, whereas radicals is a ketyl radical with an oxygen attached to the carbon on resident in the active site of R1 are difficult to observe because which the unpaired electron is localized (Table 2) (44). of saturation effects (35). In a preliminary saturation–recovery Whereas we are examining this radical structure in more detail ⅐ experiment, we observed that the T1 of the Y is strongly by using high-field electron (ENDOR) methods (26), it is clear temperature-dependent, ranging from Ϸ3msat12KtoϽ1 ␮s that this radical is not on the normal nucleotide-reduction at 55 K. The latter time approaches the length of the stimulated pathway. It represents yet another example of the difficulty of echo pulse sequence. Above 60 K, the pulse EPR spectrum of deriving mechanistic insight from radicals generated on a the Y⅐ is no longer detectable because of its fast magnetization second-to-minute time scale (1).

Table 1. EPR parameters of thiyl radicals, persulfide radicals, disulfide radical anions, and the 10-s sulfur-based R1 radical observed during reaction of E441Q R1 with R2 and CDP

Radical g1 g2 g3 a(G) Ref.

RCH2S⅐ 2.134–2.321 1.99–2.03 1.92–1.99 12–16 (H1)†,28–39 (H1)† 28–31 RCH2SS⅐ 2.052–2.062 2.023–2.027 1.998–2.006 5–11 28, 36, 37 ៮⅐ RCS2S O SCH2RЈ 2.017–2.024 2.014–2.020 2.002 8–11 (H1,H2)† 28, 38–40 This work 2.023 2.015 2.002 10 (H1,H2)*† *Parameters used in the simulation of the experimental spectrum shown in Fig. 3 F. †H1 is one proton, and H2 is the second proton. Downloaded by guest on October 2, 2021 Biochemistry: Lawrence et al. Proc. Natl. Acad. Sci. USA 96 (1999) 8983

Table 2. EPR parameters of ␣-hydroxy radicals, ketyl radicals, allowed removal of the predominant Y⅐ feature from the 10-s and the 3-min nucleotide radical formed in reaction of E441Q R1 spectrum at T Ն60 K. with R2 and CDP The value of the pulsed method, as well as its validity, is best

Radical gav Ref. illustrated with the radical species observed at longer times (3 min) and also present in the 10-s sample. The method of 2.0029–2.0032 18, 44–46 difference spectroscopy is shown in Fig. 4A. The Y⅐ spectrum is recorded at time t ϭ 0, and the spectrum of the sample at 3 min is again recorded. At T ϭ 20 K, the relaxation times of the Y⅐ are still long enough that the spin echo-detected 2.0039–2.0044 18, 44 spectrum results in a superposition of Y⅐ and the new radical. The resulting difference spectrum is also shown in Fig. 4A.By using the three-pulse echo filter at higher temperature, one can 2.0049 18, 44 obtain the spectrum of the unknown radical directly (Fig. 4B). A comparison of the two spectra (Fig. 4 A and B) reveals that they are identical. Thus, for class I RNRs, the time-domain This work 2.0051* methodology provides a new tool to facilitate identification of new radicals generated in R1 that are masked by the intensity ϭ ϭ *The determined principal g values were g1 2.0072, g2 2.0061, and ⅐ ϭ of the Y on R2. g3 2.0021. Having established the utility of the pulsed method, we DISCUSSION applied it to the 10-s radical. The results are shown in Fig. 3C. Removal of the Y⅐ by time-domain spectroscopy greatly sim- Mutant R1s, kinetics, and EPR spectroscopy have proven to be plifies the observed signal and reveals two low-field g values. effective tools to investigate the mechanism of class I RNRs Furthermore, it demonstrates that even at 10 s, a substantial (1). Both our laboratory and that of Sjo¨berg have mutated the amount of the 3-min radical is already present. The kinetic conserved glutamate residue of class I E. coli RNR (E441) in relationship between decay of the 10-s radical and formation an effort to elucidate its function (22, 23). Our mechanistic of the 3-min radical remains to be determined. rationale for this mutation is based on our proposal that the Given that the 3-min radical appears to be a single species rate-determining step in the chemistry of the nucleotide and its spectrum is well characterized (Figs. 3D and 4B), one reduction is reduction of 3 by a disulfide radical anion to can now obtain the spectrum of the 10-s radical by subtraction generate a 3Ј-deoxynucleotide radical 4 and a disulfide of the 3-min radical. The resulting spectrum (Fig. 3E) and its (Scheme 1, step 4) (10). In this mechanism, protonation of the simulation (Fig. 3F) are consistent with a disulfide radical ketone by the protonated E441 is required for the electron anion (28, 38–40). As noted in Table 1, the three principal-axis transfer to be thermodynamically favorable (10, 17). Our g values obtained are not compatible with previously reported model thus predicts that both a disulfide radical anion and 3 thiyl radicals or persulfide radicals (28–30, 36, 37). might build up in E441Q R1. Both of these species can To further characterize this species, efforts were undertaken potentially be detected experimentally. to obtain its visible spectrum by diode-array SF UV-vis We initially carried out SF UV-vis experiments with E441Q spectroscopy. The difference spectrum obtained at 10 s rela- R1 monitoring A412 to obtain insight into the rapidity and ⅐ Ϫ1 extent of Y loss. We observed an increase in A412 (0.8 s ), maximizing around 5 s, followed by a slow decrease (0.035 sϪ1). To see an increase in A412, a second species must be forming and have a larger extinction coefficient than the Y⅐ (1,800 MϪ1⅐cmϪ1), which is lost. Examination of the proposed reac- tion pathway for nucleotide reduction (Scheme 1) reveals that the only species with absorption features at Ϸ400 nm are the Y⅐ and a disulfide radical anion. Our initial studies have therefore focused on identification of the species present at 5–10 s by using visible and EPR spectroscopies. The appear- ance of this new feature in the 400-nm region on a time scale similar to that reported by Persson et al. for formation of a cysteine-based radical (27) allowed us to favor the disulfide radical anion formulation. The use of high-field (140-GHz) pulsed EPR spectroscopy has greatly facilitated our ability to identify one of the 10-s intermediates as a disulfide radical anion. The stimulated echo filter, taking advantage of the fast relaxation properties of the Y⅐ arising from its proximity to the dinuclear iron center,

FIG. 4. Stimulated echo-detected EPR spectra at 140 GHz of the E441Q R1 reaction quenched 3 min after mixing. The stimulated echo pulse sequence used was the same as in Fig. 3. The parameters (pulse spacing and repetition rates) are the same as those described in Fig. 3 for 20 K (A) and 60 K (C). (A) Spectrum at 20 K, 8 scans, collecting 300 transients per scan. The solid line displays the total signal resulting from contributions of the tyrosyl radical and the new radical. The dotted line is the spectrum of the R2 Y⅐ recorded under the same conditions. The dashed line is the spectrum of the new radical obtained by subtraction of the Y⅐ from the total signal. (B) Electron-spin echo-detected spectrum of the same mixture as A at 60 K collecting 300 transients per point, 120 scans. The g values were determined from Scheme 2. maxima, minima, and zero-crossing points of the derivative spectrum. Downloaded by guest on October 2, 2021 8984 Biochemistry: Lawrence et al. Proc. Natl. Acad. Sci. USA 96 (1999)

tive to that at t ϭ 2.5 ms (where 100% of the starting Y⅐ is 12. Gerfen, G. J., Licht, S. S., Willems, J.-P., Hoffman, B. M. & assumed to be present) is shown in Fig. 2. Further analysis Stubbe, J. (1996) J. Am. Chem. Soc. 118, 8192–8197. suggests that 20 Ϯ 5% of the starting Y⅐ is actually lost during 13. van der Donk, W. A., Yu, G., Perez, L., Sanchez, R. J. & Stubbe, the first 10 s, in agreement with the EPR data. Addition of Y⅐ J. (1998) Biochemistry 37, 6419–6426. (0.23 equivalent) back to the difference spectrum is therefore 14. van der Donk, W. A., Gerfen, G. J. & Stubbe, J. (1998) J. Am. Chem. Soc. 120, 4252–4253. required to realize the actual spectrum of the new intermedi- 15. Gerfen, G. J., van der Donk, W. A., Yu, G., McCarthy, J. R., ate, revealing a spectrum similar to those reported for disulfide Jarvi, E. T., Matthews, D. P., Farrar, C., Griffin, R. G. & Stubbe, radical anions (32, 33). Thus, the increase in A412 in the first J. (1998) J. Am. Chem. Soc. 120, 3823–3835. 5 s (Fig. 1) appears to be predominantly associated with 16. Silva, D. J., Stubbe, J., Samano, V. & Robins, M. J. (1998) formation of a disulfide radical anion (␧ ϭ 8,000–15,000 Biochemistry 37, 5528–5535. MϪ1⅐cmϪ1) which masks the loss of the Y⅐ (␧ ϭ 1,800 17. Lenz, R. & Giese, B. (1997) J. Am. Chem. Soc. 119, 2784–2794. MϪ1⅐cmϪ1). The fact that an EPR spin quantitation analysis at 18. Buley, A. L., Norman, R. O. C. & Pritchett, R. J. (1966) J. Chem. 9 GHz reveals there is not a 1:1 correspondence between loss Soc. B, 849–852. of the Y⅐ and formation of the new species suggests that the Y⅐, 19. Mao, S. S., Holler, T. P., Yu, G. X., Bollinger Jr., J. M., Booker, Biochemistry 31, disulfide radical anion, and/or other radical intermediates are S., Johnston, M. I. & Stubbe, J. (1992) 9733– 9743. being quenched. 20. Lin, A. I., Ashley, G. W. & Stubbe, J. (1987) Biochemistry 26, The detection of a disulfide radical anion is potentially 6905–6909. mechanistically significant, because our model (Scheme 1) 21. Ashley, G. W. & Stubbe, J. (1989) in Inhibitors of Ribonucleoside predicts its accumulation with E441Q R1 (10). Thus, these Diphosphate Reductase Activity, ed. Cory, J. G. & Cory, A. H. experiments provide insight into the mechanism of the reduc- (Pergamon, Oxford), pp. 55–87. tion of 3 during dNDP formation. The kinetic complexity of 22. van der Donk, W. A., Yu, G., Silva, D. J., Stubbe, J., McCarthy, the interaction of this mutant with CDP, however, requires J. R., Jarvi, E. T., Matthews, D. P., Resvick, R. J. & Wagner, E. further analysis. (1996) Biochemistry 35, 8381–8391. Finally, the paper of Persson et al. assigns the second radical 23. Persson, A. L., Eriksson, M., Katterle, B., Potsch, S., Sahlin, M. & Sjoberg, B.-M. (1997) J. Biol. Chem. 272, 31533–31541. formed on the minute time scale and already present in the 10-s 24. Salowe, S. P. & Stubbe, J. (1986) J. Bacteriol. 165, 363–366. sample as 1 (Scheme 1, step 1) (27). An examination of the 25. Bollinger Jr., J. M., Edmondson, D. E., Huynh, B. H., Filley, J., literature reveals that the gav value reported by Persson et al. Norton, J. R. & Stubbe, J. (1991) Science 253, 292–298. is inconsistent with radicals having a structural similarity to 1 26. Bennati, M., Farrar, C. T., Bryant, J. A., Inati, S. J., Weis, V., or 4 (Table 2) (18, 44–46). Although we have not studied the Gerfen, G. J., Riggs-Gelasgo, P., Stubbe, J. & Griffin, R. G. 3-min radical in-depth, we have established its principal-axis g (1999) J. Magn. Res. 138, 232–243. values (Fig. 4), which give a gav similar to that reported by 27. Persson, A. L., Sahlin, M. & Sjoberg, B.-M. (1998) J. Biol. Chem. Persson et al. A search of the literature reveals that this radical 273, 31016–31020. is most reasonably associated with a hydroxylated ketyl radical 28. Nelson, D. J., Petersen, R. L. & Symons, M. C. R. (1977) J. Chem. (Table 2; ref. 44). We propose that this radical is the 4Ј-ketyl Soc. Perkin Trans. 2, 2005–2015. 29. Akasaka, K. (1965) J. Chem. Phys. 43, 1182–1184. radical 6 (Scheme 2) generated by a thiyl radical (C462) in 30. Budzinski, E. E. & Box, H. C. (1971) J. Phys. Chem. 75, equilibrium with the disulfide radical anion. If this assignment 2564–2570. turns out to be correct, then this radical is not on the normal 31. Kou, W. W. H. & Box, H. C. (1976) J. Chem. Phys. 64, 3060–3062. reduction pathway. 32. Hoffman, M. Z. & Hayon, E. (1972) J. Am. Chem. Soc. 94, Studies with E441Q R1 have provided insight into the 7950–7957. mechanism of nucleotide reduction. A disulfide radical anion 33. Chan, P. C. & Bielski, B. H. J. (1973) J. Am. Chem. Soc. 95, has been detected by both EPR and UV-vis spectroscopies. 5504–5508. More detailed analysis is required to understand the kinetic 34. Sahlin, M., Petterson, L., Gra¨slund,A., Ehrenberg, A., Sjo¨berg, complexity of this system. However, the postulated role of B.-M. & Thelander, L. (1987) Biochemistry 26, 5541–5548. 3 35. van der Donk, W. A., Stubbe, J., Gerfen, G. J., Bellew, B. F. & E441 in the reduction of by a disulfide radical anion and the Griffin, R. G. (1995) J. Am. Chem. Soc. 117, 8908–8916. latter’s detection when E441Q R1 is studied provides support 36. Cove`s, J., Le Hir de Fallois, L., Le Pape, L., De´cout, J.-L. & for our postulated reduction mechanism of the catalytic cycle Fontecave, M. (1996) Biochemistry 35, 8595–8602. of RNR. 37. Parast, C. V., Wong, K. K., Kozarich, J. W., Peisach, J. & Magliozzo, R. S. (1995) Biochemistry 34, 5712–5717. This work was supported by National Institutes of Health Grant 38. Rao, D. N. R., Symons, M. C. R. & Stephenson, J. M. (1983) GM-29595 (J.S.) and by National Institutes of Health Grants GM- J. Chem. Soc. Perkin Trans. 2, 727–730. 38352 and RR-00995 (R.G.G.). 39. 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