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

Abstract

<i>S</i>-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 <i>S</i>-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)₂CN 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_αH-NH₂ with a CH₂ 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 ¹³C2-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 C₂=C₃ alkene substitution relative to HPPA's single bond. Interestingly, the HydG reaction with the <i>cis-p</i>-coumaric acid isomer led to the trapping of a new radical EPR signal, and EPR studies using <i>cis-p</i>-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 <i>cis-p</i>-coumaric acid. Density functional theory energetics calculations show that the <i>cis-p</i>-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.