Pyruvate formate lyase (PFL) is the paradigm of glycyl radical enzymes (GREs) and is widely distributed in anaerobic and facultative anaerobic organisms. PFL catalyzes the transformation of pyruvate into acetyl CoA and formate, connecting the glycolytic pathway with energy conservation pathways and is directly related to bacterial virulence. PFL is characterized by a post-translationally installed glycyl radical (G●), and two cysteines necessary for catalysis. G● initiate activity by activating cysteine residues by H-atom transfer and forming reactive thiyl radicals, which react with pyruvate producing a non-oxidative C-C bond cleavage. The two active site cysteines in PFL are thought to have distinct roles as thiyl radicals, but direct structural and electronic characterization of this radical chemistry has been challenging due to the instability of the radical intermediates. We employed mechanism-based inhibitors and selenocysteine substitution to gain insight into cysteinyl radical generation, kinetics and energetics. Additionally, we characterized the immune response relevant inhibition of PFL by nitric oxide.PFL reacts with the mechanism-based inhibitor methacrylate inhibiting the enzyme activity, we observed the concomitant transformation G● into a new radical specie by electron paramagnetic resonance (EPR). We assigned the new radical to a C2 tertiary methacryl radical based on spectral simulations and reactions with methacrylate isotopologues. The reaction is specific for C418 and the methacryl radical decays reforming G●. C2● is quenched, presumably by C419, exhibiting an H/D solvent isotope effect of 3.4. Acrylate also inhibits PFL irreversibly, and alkylates C418, but the acryl secondary radical is unstable and elusive to direct observation, consistent with the expected reactivity of a secondary versus tertiary carbon-centered radical. These results support the unique roles of the two active site cysteines of PFL and a C419 S−H bond dissociation energy between that of a secondary and tertiary C−H bond. We substituted the two active site cysteines of PFL for selenocysteine. Selenocysteinyl radicals are proposed to stabilize the radical chemistry long enough for direct observation. Selenocysteine incorporation reduces PFL turnover number by over 5000-fold. Although we were unable to detect selenyl radicals directly using X-band EPR, the selenocysteine mutants displayed increased solvent H/D kinetic isotope effects and changes in KM values for pyruvate and CoA, evidencing cysteinyl radical formation and the distinct roles of the two cysteines. Finally, we demonstrated that PFL is irreversible inhibited by the bacteriostatic Nitric oxide (NO) by reacting directly with G●, as demonstrated using EPR and site-directed mutagenesis. The activation of PFL by its cognate activating enzyme (PFL-AE) is also inhibited by NO by forming dinitrosyl iron complexes with the essential [Fe4S4] cluster. Whole-cell EPR and metabolic flux analyses demonstrated that PFL and PFL-AE are inhibited by NO in bacterial cell cultures, inhibiting growth and causing a metabolic shift from formate to lactate fermentation. The findings extend to class III ribonucleotide reductase (RNR), suggesting that NO targets GREs and radical SAM enzymes generally. The results implicate an immunological role of NO in inhibiting glycyl radical enzyme chemistry in the gut.