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. 2019 Jul 31;141(30):12139-12146.
doi: 10.1021/jacs.9b05926. Epub 2019 Jul 22.

The Elusive 5'-Deoxyadenosyl Radical: Captured and Characterized by Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopies

Hao Yang  1 , Elizabeth C McDaniel  2 , Stella Impano  2 , Amanda S Byer  2 , Richard J Jodts  1 , Kenichi Yokoyama  3 , William E Broderick  2 , Joan B Broderick  2 , Brian M Hoffman  1
Affiliations

The Elusive 5'-Deoxyadenosyl Radical: Captured and Characterized by Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopies

Hao Yang et al. J Am Chem Soc. .

Abstract

The 5'-deoxyadenosyl radical (5'-dAdo·) abstracts a substrate H atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, 5'-dAdo· has eluded characterization despite efforts spanning more than a half-century. Here, we report generation of 5'-dAdo· in a RS enzyme active site at 12 K using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe-4S]+ cluster to the coordinated S-adenosylmethionine (SAM) to induce homolytic S-C5' bond cleavage. We unequivocally reveal the structure of this long-sought radical species through the use of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies with isotopic labeling, complemented by density-functional computations: a planar C5' (2pπ) radical (∼70% spin occupancy); the C5'(H)2 plane is rotated by ∼37° (experiment)/39° (DFT) relative to the C5'-C4'-(C4'-H) plane, placing a C5'-H antiperiplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4'-H. The agreement between φ from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo· itself, and not determined by its environment.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 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).
Figure 2.
Figure 2.
X-band EPR spectra of the ([4Fe–4S]++SAM) PFL-AE complex (A) before and (B) after photolysis at 12 K with 450 nm LED for 1 h to produce the 5′-dAdo· radical with near-complete loss of the initial complex signal. Conversion is quantitative; residual cluster signal in spectrum B is from the enzyme out of the laser beam. (C) Time course for decay of ([4Fe–4S]++SAM) (■) upon photolysis monitored at 3600 G and increase of 5′-dAdo· (●) monitored at 3360 G. The two progress curves are fit to stretched-exponential (as the result of light scattering within the “snow” samples) decay, I = exp(–[t/τ]n), and rise, I = 1 – exp(–[t/τ]n), functions with the same parameters, τ = 11(1) min and n = 0.43(2). EPR conditions: microwave frequency, 9.38 GHz; modulation, 10 G; T, 12 K.
Figure 3.
Figure 3.
A–E, X-band EPR spectra (black) and simulations (red) of 5′-dAdo·. 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′-dAdo· with [adenosyl-13C10,15N5]-SAM; E, 5′-dAdo· with [adenosyl-5′−13C]-SAM. Features associated with the minor 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 line width at low field is attributable to g/A strain. Simulations: Generated with EasySpin using a 5′-dAdo· model with parameters listed in Table 1. EPR conditions: microwave frequency, 9.38 GHz (A–C and G) and 34 GHz (D–F); modulation, 10 G; T, 40 K. Simulations: generated with Easyspin using a 5′-dAdo· model with parameters listed in Table 1. G, Q-band CW 13C ENDOR of 5′-dAdo·. From 11 to 15 MHz, generated with [adenosyl-13C10,15N5]-SAM where (▼) represents 13C Larmor frequency and the “goalpost” connecting the doublet from 1′−13C and/or 2′−13C, split by A = 0.8 MHz (see eq 1). From 27 to 170 MHz, generated with [adenosyl-13C10,15N5]-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 and 149 MHz also seen when generated from [adenosyl-5′−13C]-SAM. It is overlaid on the signal from 1H of C4′ and 5′ in the spectrum with natural-abundance SAM (gray line). CW ENDOR conditions: microwave frequency, 34.8 GHz; modulation, 2 G; T, 2 K.
Figure 4.
Figure 4.
DFT models of 5′-dAdo·. Top: Perspective view of the optimized structure. Adenosine is represented by a violet sphere; the isosurface plot of the calculated HOMO (yellow) uses an isodensity of 0.08 au and shows the direction of g3 normal to the C5′H2 plane. Bottom: left, conformer with a dihedral “twist” at the C5′–C4′ bond, φ = 0; right, optimized structure geometry φ = 39.3°.
Figure 5.
Figure 5.
Cartoon illustrating the proposed movement of 5′-dAdo· upon S–C(5′) bond cleavage, as inferred from EPR and ENDOR data, based on PDB ID 3CB8; [–C5′(H)2] is shown as a purple ball.
See this image and copyright information in PMC

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