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

[Image: see text] S-Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound cofactor in the superfamily of radical SAM (rSAM) enzymes, in which one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic cleavage of the S-adenosyl methionine to generate the 5′-deoxyadenosyl radical (5′dAdo(•)), a potent H-atom abstractor. HydG, a member of this rSAM family, uses the 5′dAdo(•) radical to lyse its substrate, tyrosine, producing CO and CN that bind to a unique Fe site of a second HydG Fe–S cluster, ultimately producing a mononuclear organometallic Fe-l-cysteine-(CO)(2)CN complex as an intermediate in the bioassembly of the catalytic H-cluster of [Fe–Fe] hydrogenase. Here we report the use of non-native tyrosine substrate analogues to further probe the initial radical chemistry of HydG. One such non-native substrate is 4-hydroxy phenyl propanoic acid (HPPA) which lacks the amino group of tyrosine, replacing the C(α)H-NH(2) with a CH(2) at the C2 position. Electron paramagnetic resonance (EPR) studies show the generation of a strong and relatively stable radical in the HydG reaction with natural abundance and (13)C2-HPPA, with appreciable spin density localized at C2. These results led us to try parallel experiments with the more oxidized non-native substrate coumaric acid, which has a C(2)=C(3) alkene substitution relative to HPPA’s single bond. Interestingly, the HydG reaction with the cis-p-coumaric acid isomer led to the trapping of a new radical EPR signal, and EPR studies using cis-p-coumaric acid along with isotopically labeled SAM reveal that we have for the first time trapped and characterized the 5′dAdo(•) radical in an actual rSAM enzyme reaction, here by using this specific non-native substrate cis-p-coumaric acid. Density functional theory energetics calculations show that the cis-p-coumaric acid has approximately the same C–H bond dissociation free energy as 5′dAdo(•), providing a possible explanation for our ability to trap an appreciable fraction of 5′dAdo(•) in this specific rSAM reaction. The radical’s EPR line shape and its changes with SAM isotopic substitution are nearly identical to those of a 5′dAdo(•) radical recently generated by cryophotolysis of a prereduced [4Fe-4S]-SAM center in another rSAM enzyme, pyruvate formate-lyase activating enzyme, further supporting our assignment that we have indeed trapped and characterized the 5′dAdo(•) radical in a radical SAM enzymatic reaction by appropriate tuning of the relative radical free energies via the judicious selection of a non-native substrate.