The Elusive 5′-Deoxyadenosyl Radical: Captured and Characterized by EPR and ENDOR Spectroscopies

The Elusive 5′-Deoxyadenosyl Radical: Captured and Characterized by EPR and ENDOR Spectroscopies.

Hao Yang1, Elizabeth C. McDaniel2, Stella Impano2, Amanda S. Byer2, Richard J. Jodts1, Kenichi Yokoyama3, William E. Broderick2, Joan B. Broderick2*, Brian M. Hoffman1*.

1Department of Chemistry, Northwestern University, Evanston, IL 60208. 2Department of Chemistry & Bio- chemistry, Montana State University, Bozeman, MT. 59717. 3Department of Biochemistry, Duke University, Durham, NC.

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ABSTRACT: The 5¢-deoxyadenosyl radical (5¢-dAdo•) cofactor. We now know that coenzyme B12 abstracts a substrate H-atom as the first step in rad- serves as a reversible radical generator, un- ical-based transformations catalyzed by adeno- dergoing Co-C bond homolysis to give a 5’- sylcobalamin-dependent and radical S-adenosyl-L- deoxyadenosyl radical (5¢-dAdo•) that initi- methionine (RS) enzymes. Notwithstanding its cen- ¢ ates chemistry by H-atom abstraction from tral biological role, 5 -dAdo• has eluded character- 3 ization despite efforts spanning more than a half- substrate. The involvement of 5¢-dAdo• has century. Here we report generation of 5¢-dAdo• in been shown primarily through label-transfer a RS enzyme active site at 12 K, using a novel ap- studies, for example by observing incorpora- proach involving cryogenic photoinduced electron tion of substrate deuterons into adenosylco- + transfer from the [4Fe-4S] cluster to the coordi- balamin during catalysis.4-5 Important in- nated S-adenosylmethionine (SAM) to induce ho- sights into its behavior have been attained molytic S-C5¢ bond cleavage. We unequivocally re- through kinetic and spectroscopic investiga- veal the structure of this long-sought radical species through use of electron paramagnetic reso- tions in which the Co-C5¢ bond of B12 is pho- nance (EPR) and electron nuclear double reso- tolytically cleaved, after which the 5¢-dAdo• nance (ENDOR) spectroscopies with isotopic label- radical can either reform that bond or pro- ing, complemented by density-functional computa- ceed to react with substrate.6-7 However, tions: a planar C5¢ (2pπ) radical (~70% spin occu- strenuous efforts over many years8 to trap pancy); the C5¢(H)2 plane is rotated by ~ 39° relative and fully characterize the 5¢-dAdo• formed to the C5¢-C4¢-(C4¢-H) plane, placing a C5¢-H anti- from B12 have been unavailing: the radical has periplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4¢-H. simply been too reactive to trap. The significance of the 5¢-dAdo• radical in biology broadened dramatically with the The discovery by Barker in 1958 of a cobal- recognition that S-adenosylmethionine amin involved in catalyzing an isomerization (SAM) plays a role analogous to that of coen- 1 9-10 reaction, and its subsequent identification zyme B12 in initiating radical reactions. as adenosylcobalamin (coenzyme B12 (Fig 1)) This role for SAM was first suggested for ly- 11-12 by Hodgkin in 1961,2 set the stage for decades sine 2,3-aminomutase and pyruvate for- 13-14 of study focused on the biochemical reac- mate-lyase activating enzyme, but it is now tions of this remarkable organometallic implicated throughout the vast and diverse RS superfamily.15 Until recently, the accepted Fig. 1. Adenosylcobalamin/coenzyme B12 (left) and S-adenosylmethionine bound to a [4Fe-4S] cluster in the active site of RS enzymes (modeled at right) both serve as precursors to the 5¢-dAdo• radical intermediate (center). mechanism for the RS enzymes involved the Co(II; S = ½) corrinoid and the S= ½ rad- SAM coordination to the unique iron of the ical result in a broad, poorly-defined elec- [4Fe-4S] cluster in the active site; electron tron paramagnetic resonance (EPR) signal transfer from the reduced [4Fe-4S]+ cluster to that is not amenable to analysis.21 A sharp SAM promoted reductive cleavage of SAM to fluid-solution FT-EPR spectrum acquired give methionine and 5¢-dAdo•, which initi- upon photolysis of B12 and thought to arise ates chemistry via substrate H-atom abstrac- from 5¢-dAdo•, is instead the result of rapid tion.15-16 The recent discovery of the catalyti- radical rearrangement rather than 5¢-dAdo• cally competent organometallic intermediate itself (see SI Text, Fig. S2).22 An organic rad- W formed during the RS reaction provides a ical that accumulates during reaction of the new twist on the mechanism, by implicating RS enzyme spore photoproduct lyase with a coenzyme B12-like organometallic interme- SAM at 40 ˚C also has been proposed to be diate with an Fe-C5¢ bond that undergoes ho- 5¢-dAdo•,23 but we show in the SI that this molytic bond cleavage to release 5¢-dAdo•.16- radical too is not 5¢-dAdo• (see SI Text, Fig 18 S3, Table S1). Insights into the characteristics of the 5¢- Here, after more than half a century of ef- dAdo• radical intermediate in RS enzymes forts by numerous investigators to trap and have been achieved via the SAM analog 3¢,4¢- characterize the biologically central 5¢- anhydro-S-adenosylmethionine (anSAM), dAdo• radical, we demonstrate the for- which generates an allylically stabilized 5¢- mation of 5¢-dAdo• in a RS enzyme active dAdo• analog that is amenable to character- site, by using a novel approach involving ization,19-20 but so far attempts to generate photoinitiated electron transfer from the re- and characterize the 5¢-dAdo• radical itself duced [4Fe-4S]+ cluster to the coordinated have not been productive. For example, pho- SAM. The resulting 5¢-dAdo• is definitively tolysis of coenzyme B12 appears to produce 5¢- identified through the use of isotopically la- dAdo•, but spin-spin interactions between beled SAM combined with EPR and electron-nuclear double resonance Photolysis was carried out in situ using a 450 (ENDOR) spectroscopy, with its structure an- nm Thorlabs diode laser. The time course of alyzed using density functional theory (DFT) 12 K photolysis of (PFL-AE1++SAM) complex computation. was monitored during intra-cavity photolysis in an X-band EPR spectrometer. The in situ Experimental Methods photolysis of the (PFL-AE1++SAM) complex Materials pre-reduced to the [4Fe-4S]+ state using ei- 13 15 [Methyl- C]-L-methionine, N-L-methio- ther deazariboflavin or dithionite gives iden- nine, and 2,8-D2-1¢,2¢,3¢,4¢,5¢,5¢¢-D6-adenosine tical EPR spectra, Figure S1. The in situ pho- 5¢-triphosphate salt solution were purchased tolyzed samples were subjected to X- and Q- from Cambridge Isotope Laboratories, Inc. band CW EPR and Q-band ENDOR meas- 13 15 [ C10, N5]-adenosine 5¢-triphosphate sodium urements as described below. salt solution was purchased from Sigma and EPR and ENDOR Measurements 3,3,4,4-d4-L-methionine was obtained from X-band CW EPR spectroscopy was con- CDN Isotopes. ducted on a Bruker ESP 300 spectrometer Protein and SAM Preparation equipped with an Oxford Instruments ESR PFL-AE, unlabeled SAM, and labeled 910, while Q-band CW EPR spectroscopy SAMs were prepared as previously re- was conducted on a Bruker EMX spectrom- 24 ported. eter equipped with an Oxford Instruments Photolysis Mercury iTC continuous helium flow cryo- Pyruvate formate-lyase (PFL-AE) was pre- stat. Typical experimental parameters were at pared for photolysis by reducing the enzyme 12 K and 40 K, 9.38 GHz or 34.0 GHz, and 10 and adding labeled or unlabeled SAM. Pho- G modulation amplitude. EPR simulations 5.2.23 toreduced PFL-AE was prepared as previ- were performed with the EasySpin pro- 25 ously described24 with minor alterations. gram operating in Matlab. PFL-AE (0.55 mM), dithiothreitol (DTT, 1.0 35 GHz CW and pulse ENDOR spectro- mM) and deazariboflavin (200 µM, dissolved scopic data were collected on spectrometers, in DMSO) were combined in buffer (50 mM described previously,26-28 that are equipped Tris, 100 mM KCl, pH 7 .5) in an anaerobic with liquid helium immersion dewars for COY chamber. The sample was irradiated by measurements at 2 K. The CW measure- a 500 W halogen lamp for 1 h while the sam- ments employed 100 kHz field modulation ples remained in an ice water bath. Labeled and dispersion-mode detection under rapid or unlabeled SAM (5.5 mM) was then added. passage conditions. 1H CW ENDOR spectra The samples were then transferred to EPR employed bandwidth broadening of the RF tubes and frozen in liquid nitrogen inside to 100 kHz to improve signal-to-noise.29 1H the COY chamber. CW ENDOR spectra were collected using Dithionite reductions were performed sim- the stochastic-field modulation detected 30 ilarly, as follows: PFL-AE (0.55 mM), DTT (1.0 ENDOR sequence, to improve ENDOR line mM), and sodium dithionite (3.0 mM) were shape. For a single molecular orientation and 1 13 combined in an anaerobic COY chamber for nuclei with nuclear spin of I = /2 ( C), the 1 and allowed to sit 3 min before SAM (5.5 mM) ENDOR transitions for the ms = ± /2 electron was added. The samples were flash frozen as manifolds are observed, to first order as described above and stored in liquid nitro- shown in equation: gen.

A intermediate in RS enzymes could be uu=± (eq. 1) ± n 2 cleaved photolytically, similar to the Co-C5¢ bond of coenzyme-B12. If this were to occur,

where un is the nuclear Larmor frequency, and A is the orientation-dependent hyperfine coupling. When un > A/2, the pattern is a doublet split by A and centered at un; when un < A/2, the pattern is a doublet centered at

A/2 and split by 2un. DFT calculations All DFT computations of 5′-dAdo• were performed in ORCA 4.0.1.31 The initial coor- dinates for the 5¢-dAdo fragment were taken + from a 1.5 Å resolution crystal structure of a Fig. 2. X-band EPR spectra of (A) ([4Fe-4S] +SAM) PFL-AE complex before and (B) after photolysis at radical-SAM methyltransferase with SAM 12K with 450 nm LED for 1 hr to produce the 5¢- bound to the [4Fe-4S] cluster (PDB 3DLC). dAdo• radical with near-complete loss of the ini- All protein residues, water, and other mole- tial complex signal. Conversion is quantitative; re- cules were removed and the adenine was re- sidual cluster signal in spectrum B is from enzyme placed with a methyl group. Hydrogens were out of the laser beam. (C) Time course for decay of ([4Fe-4S]++SAM) (▪) upon photolysis monitored at added appropriately to the structure with the 3600 G, and increase of 5¢-dAdo• (•) monitored at starting geometry of the 5¢-C hydrogens be- 3360 G. The two progress curves are fit to ing in a pseudo-tetrahedral conformation. stretched-exponential (as the result of light scatter- Both geometry optimizations and single- ing within the ‘snow’ samples) decay, I = exp(-[t/t]n), n point calculations used the spin unrestricted and rise, I = 1-exp(-[t/t] ), functions with the same parameters, t =8±1 min and n = 0.52±0.02. EPR B3LYP32-34 hybrid functional and the Ahl- conditions: microwave frequency, 9.38 GHz; modu- richs’ valence triple-x with a polarization lation 10 G; T = 12 K. function basis set.35 The molecular orbitals were visualized as gaussian cubes and an then photoinitiated homolysis of W would isosurface of 0.08 au in Pymol. Hyperfine produce the diamagnetic [4Fe-4S]2+ cluster and g tensors were calculated by the cou- and the sought-after 5¢-dAdo• radical. How- pled−perturbed self-consistent field (SCF) ever, because SAM has been reported to be approach as implemented in ORCA 4.0 using photochemically reactive,39-40 we first carried the B3LYP hybrid functional and EPR-III out control experiments in which SAM and basis,36-37 in combination with the accurate PFL-AE/[4Fe-4S]1+ were combined and pho- spin-orbit coupling operator [RI- tolyzed in the absence of substrate PFL, cir- SOMF(1X)].38 Calculations using the BP8632 cumstances in which SAM is not enzymati- functional were carried out in parallel. Table cally cleaved and W does not form. Much to S2 compares the similar results for the two our surprise, irradiation of such samples in functionals. the EPR cavity at 450 nm and 12 K results in rapid conversion of the SAM-bound [4Fe- 4S]+ state of PFL-AE (Fig. 2A) to one with a Results new free-radical signal that is partially satu- + Photolysis of PFL-AE/[4Fe-4S] /SAM. rated and thus poorly-resolved at 12 K, Fig. The present study was indirectly prompted 2B, but becomes well-resolved at 40 K (Fig. by the idea that the Fe-C5¢ bond of the W17-18

3A). A time course of the photolysis shows are possible. Rearrangement to form a 4′ rad- the loss of the [4Fe-4S]1+ signal directly corre- ical, or even a cyclo-adenosine radical must lates with the appearance of the new radical be considered, and if the highly reactive 5′- signal (Fig. 2C). dAdo• is formed, it might have attacked a The data is consistent with photoinduced nearby protein residue, to generate a protein electron transfer (ET) from the [4Fe-4S]+ radical, even at 12 K. We therefore pursued cluster to SAM,41 to provide the EPR-silent detailed studies that unequivocally identify [4Fe-4S]2+ cluster and a SAM-derived organic the structure of this radical species. radical cryo-trapped for study; such photoin- The radical as trapped in the active site of duced ET has not previously been reported PFL-AE is stable at 77K and below, but is lost for RS enzymes. Based on known RS enzyme upon annealing for one minute at 150K. Fig- chemistry, the most likely identity of the new ure 3A shows that the EPR spectrum of the radical species would be the 5¢-dAdo• radical radical species under nonsaturating condi- resulting from reductive cleavage of SAM. tions (40 K) can be well simulated by incor- However, other forms for the trapped radical porating 1H hyperfine interactions J

Figure 3. Figure 3 A-E, X-band EPR Spectra (black) & Simulations (red) of 5¢-dAdo•. A, 5¢-dAdo• generated with: A natural abundant SAM; B, [adenosyl-5¢,5¢¢-D2]-SAM; C [adenosyl-2,8-D2-1¢,2¢,3¢,4¢,5¢,5¢¢-D6]-SAM; D, 5¢- 13 15 13 dAdo• with [adenosyl- C10, N5]-SAM; E, 5¢-dAdo• with [adenosyl-5¢- C]-SAM. Features associated with minor- ity photolysis products are revealed in spectra of isotopologues (B, C) and indicated by (*) F, Q-band EPR, 5¢-dAdo• generated with natural abundant SAM; the additional linewidth at low field attributable to g/A strain. Simulations: Generated with EasySpin26 using a 5¢-dAdo• model with parameters listed in Table 1. Con- ditions: EPR, microwave frequency 9.38 GHz (A-C, G and H), and 34 GHz (D-F); modulation, 10 G; T = 40 K. Simulations: Generated with Easyspin26 using a 5¢-dAdo• model with parameters listed in Table 1. G, Q-band 13 13 15 CW C ENDOR of 5¢-dAdo•. From 11- 15 MHz, generated with [adenosyl- C10, N5]-SAM where (▼) represents 13C Larmor frequency and ‘goalpost’ connecting the doublet from 1′-13C and/or 2′-13C, split by A = 0.8 MHz (see 13 15 eq S1). From 27-170 MHz, generated with [adenosyl- C10, N5]-SAM where (●) represents A/2 and only the high- frequency members of the doublets for 3′-13C and 5′-13C are seen, as indicated, separated from their respective A/2 by 13C Larmor frequency. Spectrum between 70-149 MHz also seen when generated from [adenosyl-5¢-13C]- SAM. It is overlaid on signal from 1H of C4′ and 5′ in spectrum with natural-abundance SAM. CW ENDOR Conditions: microwave frequency 34.8 GHz; modulation, 2 G; T = 2 K. appropriate for trapped 5¢-dAdo•, Fig 3A. isotropic splitting from the 4′-Cβ proton. The simulation25 includes anisotropic hyper- This splitting in turn is lost in both the X- fine couplings to two near-equivalent a-type band and Q-band spectra of the radical gen- 1H on 5¢-C plus coupling to one near-iso- erated with the perdeuterated-Ado SAM 1 tropic b-type H on 4¢-C; the hyperfine ten- isotopologue ([adenosyl-2,8-D2- sors are reported in Table 1. The two result- 1¢,2¢,3¢,4¢,5¢,5¢¢-D6]-SAM) (Fig. 3C and S5). To- ing a-type 1H hyperfine tensor components, gether, these measurements show that the confirmed and refined as described below, radical formed by photolysis is indeed the have values roughly in the ratio, A1/A2/A3 ~ long-sought 5′-dAdo•. 1/2/3, as expected for a trigonal-planar, Ca-1H This identification is further confirmed carbon radical, and indeed quantitatively and the characterization of 5¢-dAdo• is agree quite well with those of the Ca-1H rad- strengthened and enriched by examination 13 ical of •CH(COOH)2 formed by irradiation of of the radical formed with C isotopologues. 13 15 malonic acid (Table 1). In addition, it is well- When prepared with [adenosyl- C10, N5]- established 42-44 that a b-type 1H, such as the SAM (Fig 3D), the radical X-band EPR spec- 1H-4¢-C of 5¢-dAdo•, gives a large, near-iso- trum shows additional splittings from two tropic coupling, as observed here (Table 1). 13C, one with the anisotropic hyperfine ten- The Q-band EPR spectrum (Fig. 3F) is sor expected42-44 for the 5¢-13Cα ‘sp2’ carbon equally well simulated with these parame- and one with the smaller, nearly isotropic ters, and has the additional benefit of en- coupling expected for the 4¢-13Cβ carbon (Ta- hancing the influence of the small free-radi- ble 1). The assignment of the two couplings cal g-anisotropy, which helped refine both to their respective carbons is confirmed by the g and hyperfine tensors (Table 1). Of par- the EPR spectrum of radical prepared from ticular note as an ‘internal check’, the inter- singly-labeled 5¢-13C SAM, (Fig 3E) which mediate hyperfine component of an α-pro- shows the splittings with the anisotropic ten- ton must lie parallel to the 2pπ orbital, which sor assigned to 5¢-13Cα (Table 1). Note that 42-46 13 is in turn parallel to g3≈ ge, and thus must the maximum C coupling for a spin in a car- 42- be parallel for the two α-protons of 5¢-dAdo•. bon 2pπ orbital must also lie along g3 = ge, This was required for the optimized simula- 44 as found experimentally for 5¢-13C, without tions of the radical spectra, particularly the prior constraint (Table 1). Q-band spectrum (Table 1), even though the The Q-band ENDOR spectra for 5¢-13C- tensors were determined without imposing SAM, Fig. 3G, shows a peak centered at a fre- this as a prior constraint. quency corresponding to approximately A3/2 Confirmation of Radical Species using SAM Isotopologs of 5¢-13C (Table 1). The Q-band ENDOR To confirm this new radical species as the spectra from the radical prepared from [ade- 1 13 15 5¢-dAdo• radical, and to refine its H hyper- nosyl- C10, N5]-SAM (Fig. 3G) show not only fine interaction parameters, we carried out the 5¢-13C signal, but also a signal from the photolysis experiments utilizing isotopically near-isotropically coupled 3¢-13C centered at labeled SAMs.24 Assignment of the a-type a frequency corresponding to essentially half couplings to the two 5¢-Cα protons is con- its isotropic coupling (Table 1), as well as a firmed by the collapse of both the X-band doublet with a much weaker coupling (A = (Fig. 3B) and Q-band (Fig. S5) spectra of the 0.8 MHz) assigned to 1¢-13C and/or 2¢-13C; the 13 1 radical generated with [adenosyl-5¢,5¢¢-D2]- signal from 4¢- C is hidden under the H SAM to a doublet resulting from the near- ENDOR response from weakly coupled

protons (Fig. S6). These 13C measurements 20% 12C, and for E is the sum of 85% 13C 5¢-dAdo• 12 unambiguously confirm: the photochemi- and 15% C. cally generated radical is indeed, 5¢-dAdo•. b Hyperfine tensors in parentheses are from DFT calculation with B3LYP/G (SI). The energy-minimized structure (Fig 4) has equivalent C5′-H bonds Table 1. Hyperfine tensors (MHz) of 5¢-dAdo• from and 1H hyperfine tensors, whereas experiment gives experimenta plus DFT-computed,b with reference slightly different tensors for the two. We attribute values for radicals generated from malonic acidc the observed inequivalence to a slight desymmetri- zation due to interactions in the enzyme active site 5′-dAdo• A1 A2 A3 that are absent in the computation. 1 5′-C- Haa -15(-28) -105(-104) -60(-63) c 1 13 H and C tensors for •CH(COOH)2 formed by 1 5′-C- Hab -20(-25) -95(-101) -60(-61) irradiation of malonic acid, taken from McConnell 47 48 1 4′-C-1Hβ +80(+90) +80(+90) +110(+105) et al, and Cole and Heller respectively. H tensors for •CH2(COOH) (g = [2.0042, 2.0034, 2.0020], 5′-13Ca +10(+2) +10(+3) +230(+240) gave = 2.0032) formed by irradiation of malonic acid, 4′-13C 60(+69) 80(+86) 60(+68) see formed by irradiation of malonic acid, see ref 46. 3′-13C 40(-36) 40(-33) 50(-37) The somewhat greater inequivalence between the hyperfine tensors for two a-type 1H, of 5¢-dAdo• 1′-or 2′-13C 0.8 0.8 3 and •CH2(COOH) likely reflect influence of con- c •CH(COOH)2 A1 A2 A3 tacts with the surrounding residues, as does the ob- 1Ha -91 -29 -58 servation that the two radicals have the same gave, yet differ in their in-plane g-values, g , g . 13Ca +22.8 +42.2 +212.7 1 2 c •CH2(COOH) A1 A2 A3

1 Haa -30 -55 -91 1 Hab -37 -59 -92 Structure of 5¢-dAdo• a Signs (+/-) of experimental 5¢-dAdo• tensors are Beyond the identification of 5¢-dAdo•, the assigned in correspondence to ref 44; the hyperfine observed hyperfine interactions teach us sign for 1¢- and/or 2¢-13C, 3¢-13C, and 4¢-13C are not about its electronic and geometric structure. determined. The simulations employ, g = [2.0075, The two 1Hα and the 5¢-13Cα coupling tensors 2.0015, 2.000]; gave = 2.003 and tabulated hyperfine 1 1 are in good agreement with those for the odd couplings. The A3 components for Haa and Hab electron in the 2pπ orbital of the planar (sp2) are parallel to g3, normal to the C5’H2 plane (see Fig 46 47 1 3) as seen in refs & . For Haa, A1|| g1; simulation carbon-centered radical of x-irradiated ma- 1 46 required the Hab tensor be rotated relative to that lonic acid (Table 1). The match, notably in 1 0 of Haa by an angle approaching a ~120 expected the isotropic couplings, indicates that the 2 ′ for sp hybridized C5 and found by DFT optimiza- spin density in the 2pπ orbital on C5¢of 5¢- tion, but because of the small g-anisotropy this angle could be allowed to vary over the range, 900 ≲ dAdo• (ρπ) is comparable to that in malonic 0 1 0 a ≲ 120 . A3 for 4′-C- Hβ makes an angle of ~40 with acid, ρπ ~ 0.7. Of particular note, the spin-po-

respect to g3. The EPR simulations tightly constrain larization-induced isotropic coupling of the the values of the tensor components; for strongly- 2pπ spin of planar 13C5¢ would have been ′ ′ 13 coupled nuclei (all but 1 and/or 2 - C), numerous sharply increased by a pseudo-tetrahedral simulations indicate that one tensor component value may vary by as much as ±5 MHz, but the sum ‘doming’ distortion at C5¢, which would in- of multiple variations must also be no more than troduce 2s character, with its large isotropic roughly ±5 MHz. Additional EasySpin simulation pa- coupling,43 into the odd-electron orbital. rameters: EPR linewidth: 36 MHz (X-band); 45 MHz Thus we infer that the H2-C5¢-C4¢ fragment is (Q-band). Hyperfine strain parameters employed to essentially planar. The isotropic coupling of account for unresolved hyperfine coupling: [30, 10, 10] MHz for A - C, E, F; [30, 10, 40] MHz for D. The a proton b to a carbon 2pπ electron spin, such simulation for D is the sum of 80% 13C 5¢-dAdo• and as 1H-C4¢, is known to obey the relationship,

2 aiso ≈ ρπBcos φ MHz, where ρπB ≈ 140 MHz, agreement with the experimental analysis. and φ is the dihedral angle between the 2pπ The computations further generate the radi- orbital and the Cb-H bond,43 corresponding cal’s structure (Fig. 4, top), which exhibits to the angle between the [4¢CH-4¢C-5¢C] both a rigorously planar geometry at C5¢ and plane and of the 2pπ orbital (normal to the a dihedral ‘twist’ at the C5¢-C4¢ bond, as in- plane formed by C5¢ and its two H-atoms). ferred above. The dihedral (twist) angle in the Applying this relationship to the measured energy-minimized geometry φ ≈ 39o (Fig. 4, 1H-C4¢ isotropic coupling (Table 1) indicates bottom) is quite close to that indicated by the φ ≈ 37o. experimental analysis above, and creates a These inferences from the experimental structure with a C5¢-H antiperiplanar to the finding are confirmed and extended by DFT oxygen of the ribose ring, which helps stabi- computations31 for 5¢-dAdo• (SI, Table S2, lize the radical against elimination of 4¢-H, as 49 Fig. S4). Firstly, the computations yield an suggested long ago. energy-minimized structure that reproduces 5¢-dAdo• in the Active Site the observed hyperfine couplings extremely Expanding our focus, the use of additional well, Table 1, with a value for the spin den- SAM isotopologues gives a sense of the rela- sity in the C5¢ 2pπ orbital of ρπ = 0.7, in tionship of the SAM fragments (5¢-dAdo• and Met) subsequent to homolysis. When 5¢- g3 dAdo• is prepared from [3,3,4,4-methionine-

D4]-SAM, subtle changes in the shape of the EPR spectrum (Fig. S7) indicate that the ap- parent EPR linewidth of the natural-abundance spectrum includes the effects of weak hyperfine couplings to protons of the methionine sidechain. In contrast, there are no changes in the EPR spectrum when pre- 13 pared from CD3-methyl or C-methyl methionine SAM and no weakly-coupled 13C ENDOR signal is introduced by the 13C sub- stitution (Fig. S7), indicating the radical site is remote from the methyl group. These ob- servations indicate that upon SAM S-C5¢ ho- molytic bond cleavage, the C5¢ radical has shifted toward the methionine C3 and C4 and away from the methyl, as illustrated schematically in Fig 5. Such a movement leaves the spin-bearing C5¢ in proximity to the methionine sidechain, allowing unre- Figure 4. DFT models of 5¢-dAdo•: Upper: Per- solved hyperfine couplings to those hydro- spective view of optimized structure; adenosine represented by violet sphere, isosurface plot of the gens to influence the EPR spectrum, yet re- 13 calculated HOMO (yellow) using an isodensity of moved enough from the methionine C-me- 0.08 au, and showing the direction of g3, normal to thyl so as to leave no detectable couplings to ¢ the C5 H2 plane. Lower: left, conformer with di- that nucleus either in EPR or ENDOR spec- ¢ ¢ j hedral ‘twist’ at the C5 -C4 bond, = 0; right, op- tra. timized structure geometry, j = 39.3o.

Discussion The 5¢-dAdo• radical is the central species responsible for H-atom abstraction from substrate in both coenzyme-B12 and the large RS superfamily of enzymes, but until now it had eluded characterization. In the present work, we generated the 5¢-dAdo• radical in PFL-AE by cryogenic photoinduced electron transfer (ET) from the [4Fe-4S]1+ to SAM, causing reductive cleavage of SAM to generate the 5¢-dAdo• radical, and methionine chelated to the diamagnetic [4Fe-4S]2+ cluster. Because the cluster is diamagnetic, there are no spin-spin interactions that would in- terfere with characterization of the photo- Figure 5. Cartoon illustrating proposed movement generated radical, and its assignment as 5¢- of 5¢-dAdo• upon S-C(5¢) bond cleavage, as in- dAdo• has been unequivocally established ferred from EPR and ENDOR data, based on PDB 13 2 through the use of C and H labels. ID 3cb8; [-C5¢(H)2] shown as purple ball. Our early ENDOR studies showed that in other words, the enzymatic turnover system the absence of PFL substrate, SAM is coor- appears designed to avoid producing free 1+ dinated to the reduced PFL-AE [4Fe-4S] dAdo•, but rather to form the more stable 5¢- cluster through the amino acid group of dAdo• precursor W. As we have previously SAM, with the sulfonium in close contact to postulated,19 the highly reactive 5¢-dAdo• is 50-51 the cluster (<4 Å). Thus, this photoin- never free under normal catalytic conditions. duced ET is analogous to the inner-sphere An additional consideration is the nature ET thought to occur during enzymatic catal- of the process of electron transfer and SAM- ysis,50, 52 with key roles being played by con- cleavage itself. During catalysis, RS enzymes figurational interaction between donor and must overcome a large potential barrier for acceptor orbitals on the sulfonium and the reductive cleavage of SAM, due to the mis- PFL-AE/[4Fe-4S]1+ cluster,53 conformational match in reduction potentials between RS and electronic influences of substrate bind- [4Fe-4S]1+ clusters (-400 to -600 mV)55-59 and ing,54 and the mechanistic requirement of W SAM (estimated as ~ -1.8 V).60-61 The binding formation.16-18 Why, then, is 5¢-dAdo•, rather of SAM and substrate in the active site are W than , produced in the current thought to perturb both potentials, lowering W experiments? One possible answer is that the barrier to reductive SAM cleavage.54 RS formation might require the conformational catalysis via formation of W, likely also lowers consequences of the presence of substrate. the barrier to this cleavage through the sta- Unfortunately, we have not yet been able to bilization contributed by Fe-C5′ bond for- test this hypothesis because adding substrate mation, thus providing a favorable alterna- to the PFL-AE/[4Fe-4S]1+/SAM complex 52 tive pathway to direct SAM cleavage by sim- leads to immediate substrate turnover. Even ple ET to form 5′-dAdo•.16-18 when PFL-AE/[4Fe-4S]1+, SAM, and substrate are combined in rapid-freeze quench The photoinduced ET reported here instead uses photon energy to overcome the experiments with short deadtimes, W is barrier to direct formation of 5′-dAdo•. formed and 5¢-dAdo• is not observed. In

While understanding the precise nature of charge on the ACS Publications website. this process will require further study, prior 62 studies of photoreduction by metal centers AUTHOR INFORMATION suggest that in the present study the photon Corresponding Author excites the cluster to a LMCT charge transfer *Correspondence to: [email protected] excited state, which quickly relaxes to a rela- (J.B.B.); [email protected] (B.M.H. 1+ tively long-lived [4Fe-4S] ligand field excited Funding Sources state that is a potent and effective electron No competing financial interests have been declared. donor. In short, the energy of the incident This work was funded by the NIH (GM 111097 to BMH; photon, as captured in an ET-reactive excited GM 54608 to JBB). state, is sufficient to enable the direct formation of 5’-dAdo• by S-C5’ homolysis ACKNOWLEDGMENT without formation of W. This work was funded by the NIH (GM 111097 to BMH; Conclusion GM 54608 to JBB).

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NH2 H2N N N N N N N N N O O H H H H H OH OH OH S S+ OH

O 1+ O 2+ O O N N H2

TOC Graphic