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

Abstract

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)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 CαH-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 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.

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 catalytic H-cluster of [FeFe] hydrogenase: the roles of the Fe–S maturase proteins HydE, HydF, and HydG

“…The X-ray structure of a related rSAM tryptophan lyase NosL included a tryptophan oriented to favor 5 0 dAdoc H-atom abstraction from this amino acid's amino group, and the authors suggested that a similar amino H-atom abstraction drives the tyrosine fragmentation in ThiH and HydG. 45 Sayler et al 46 studied the HydG reaction with a non-native substrate, 4hydroxy phenyl propanoic acid (HPPA) ( Fig. 3B), where a simple C(2)H 2 replaces the C a H-NH 2 of tyrosine.…”

“…Thus it is clear it is the amino group of the tyrosine that is the target of the H-atom abstraction by 5 0 dAdoc, generating a transient, and yet to be observed, nitrogen centered tyrosine radical that rapidly fragments to form the DHG and 4-oxidobenzyl radical products of the rSAM component of HydG. Interestingly, when the more oxidized cis-p-coumaric acid was employed in place of HPPA, Sayler et al 46 were able to trap the primary 5 0 dAdoc which is accumulated because it cannot abstract the hydrogen atom from the sp 2 -carbon.…”

The Key Intermediate in Radical S-Adenosyl-methionine Catalysis: Caught in the Act

“…In this issue of ACS Central Science , Sayler and co-workers disclose the direct trapping and characterization of the elusive 5′dAdo· radical within the confines of the hydrogenase-cofactor maturase enzyme HydG. 2…”

“…Tyrosine lysis affords diatomic CO and CN – ligands that are components of the H cluster of di-iron hydrogenases. (B) Freeze quenching assay mixtures of HydG with the non-native substrates explored by Sayler 2 yield distinct EPR signals (right) that are assigned to the corresponding radical species. EPR signals are reproduced with permission from ref (2).…”