Pyruvate formate-lyase activating enzyme: The catalytically active 5′-deoxyadenosyl radical caught in the act of H-atom abstraction

Enzymes of the radical S-adenosyl-l-methionine (radical SAM, RS) superfamily, the largest in nature, catalyze remarkably diverse reactions initiated by H-atom abstraction. Glycyl radical enzyme activating enzymes (GRE-AEs) are a growing class of RS enzymes that generate the catalytically essential glycyl radical of GREs, which in turn catalyze essential reactions in anaerobic metabolism. Here, we probe the reaction of the GRE-AE pyruvate formate-lyase activating enzyme (PFL-AE) with the peptide substrate RVSG(734)YAV, which mimics the site of glycyl radical formation on the native substrate, pyruvate formate-lyase. Time-resolved freeze-quench electron paramagnetic resonance spectroscopy shows that at short mixing times reduced PFL-AE + SAM reacts with RVSG(734)YAV to form the central organometallic intermediate, Omega, in which the adenosyl 5 ' C is covalently bound to the unique iron of the [4Fe-4S] cluster. Freeze-trapping the reaction at longer times reveals the formation of the peptide G(734)center dot glycyl radical product. Of central importance, freeze-quenching at intermediate times reveals that the conversion of Omega to peptide glycyl radical is not concerted. Instead, homolysis of the Omega Fe-C5 ' bond generates the nominally "free" 5 '-dAdo center dot radical, which is captured here by freeze-trapping. During cryoannealing at 77 K, the 5 '-dAdo center dot directly abstracts an H-atom from the peptide to generate the G(734)center dot peptide radical trapped in the PFL-AE active site. These observations reveal the 5 '-dAdo center dot radical to be a well-defined intermediate, caught in the act of substrate H-atom abstraction, providing new insights into the mechanistic steps of radical initiation by RS enzymes.