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

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.

Trapping and Electron Paramagnetic Resonance Characterization of the 5′dAdo• Radical in a Radical S-Adenosyl Methionine Enzyme Reaction with a Non-Native Substrate

“…We note that a prior assignment of 5′dAdo • trapped in the C140A mutant of the radical SAM enzyme (RSE) spore photoproduct lyase (SPL) 34 lacked the specific magnetic isotope data needed to definitively assign the EPR spectrum. In fact, this spectrum is distinctly different from our isotope-verified 5′dAdo • radical shown here, or that of the 5′dAdo • radical generated by cryophotolysis, 4 but its approximate 1:4:6:4:1 intensity ratio of the EPR spectrum and the corresponding 1 H HFI gives an EPR signal that matches closely those found in gamma-irradiated crystals of derivatized l -alanine, 35−37 suggesting that the reaction in this Cys-to-Ala mutant of SPL could lead instead to the formation of an alanine radical at position 140. Similarly, Downs and co-workers trapped a peptide backbone radical when initiating the radical SAM reaction in ThiC in the absence of substrate.…”

“…Now we have the isotopically verified 5′dAdo • generated by low-temperature photolysis of the prereduced [4Fe-4S]-SAM cluster of the rSAM enzyme pyruvate formate-lyase, along with its attendant EPR/ENDOR/DFT characterization. 4 The CW EPR line shape of our 5′dAdo • radical produced by the HydG reaction with cis-p -coumaric acid is remarkably similar to this cryogenerated radical signal. Any small differences are likely attributable to differences in the C4′-H hyperfine coupling due to different dihedral angles (θ in our notation), no doubt resulting from the two different radical generation protocols.…”

“…Moreover, the spectral changes induced by using various isotopically labeled SAMs in both set of experiments point to the same assignment of a 5′dAdo • radical, with the vast majority spin density at the C5′ carbon, even though the specifics of the exact isotope labels are different between the two groups. As clearly described by Yang et al, 4 trapping and characterization of the “elusive” 5′dAdo • have been a long time coming, and it is interesting that it can now be done by two different approaches, the cryoillumination method and the rSAM reaction with a non-native substrate, with essentially identical 5′dAdo • radicals resulting. Our approach, trapping the 5′dAdo • radical by altering the thermodynamics of its primary H-atom abstraction target using non-native substrates, should be a broadly applicable approach toward generating the 5′dAdo • radical in other rSAM enzymes, providing a nuanced probe of its conformation and electronic structure in different enzyme environments.…”

Mechanism of Diol Dehydration by a Promiscuous Radical‐SAM Enzyme Homologue of the Antiviral Enzyme Viperin (RSAD2)

“…Human viperin is shown to be a member of the radical‐SAM superfamily of enzymes, which catalyse the transformation of a substrate by using a common catalytic step (Figure A): a highly conserved [4 Fe−4 S] cluster, which is coordinated to three cysteine residues of a conserved CxxxCxxC motif, cleaves SAM in a one‐electron reduction step to generate a 5′‐deoxyadenosyl radical (5′‐dAdo radical) intermediate . The 5′‐dAdo radical has recently been trapped and characterised, and spectroscopic studies have provided evidence for the formation of an organometallic intermediate with a Fe‐[5′‐C]‐deoxyadenosyl bond (Figure A) . The organometallic or the 5′‐dAdo radical intermediate initiates catalytic transformation by abstracting a hydrogen atom from a substrate, releasing 5′‐deoxyadenosine (5′‐dA), and generating a substrate–radical intermediate that undergoes complex rearrangement reactions to form a product .…”

Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes

“…Upon addition of SAM, the EPR spectrum was modified with novel g tensor values of [2.03, 1.92, 1.89]. This new rhombic g-tensor is indicative of the interaction between SAM and the radical SAM [4Fe-4S] cluster as shown for PFL-AE and other radical SAM enzymes (38,40,49). To better characterize the iron-sulfur clusters, two mutants were generated.…”