LAUSR

The dimeric glycyl radical enzyme pyruvate formate‐lyase (PflB; formate acetyltransferase 1) cleaves pyruvate with hypothetical half‐site reactivity to formate and acetyl‐CoA. The radical introduced onto residue G734 of PflB is transiently transferred to C419 of an adjacent cysteine pair (C418/C419) during catalysis, but it is unclear whether glycyl radical formation is dependent on C419 in vivo. We show here that a deficiency in formate production of an Escherichia coli strain synthesizing a PflBG734A variant, but lacking the autonomous glycyl radical cofactor, GrcA, could be restored by reintroducing plasmid‐encoded native PflB, but not by a PflBC418A/C419A variant, indicating that PflBC418A/C419A cannot replace GrcA. Oxygen‐dependent polypeptide cleavage of PflBC418A/C419A indicated stable glycyl radical incorporation; however, these data did not support half‐site reactivity. These in vivo findings demonstrate that glycyl radical formation is independent of subsequent radical transfer from G734 to C419, which occurs intramolecularly.