Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (Pn) leads to cleavage of the polypeptide backbone between N-C alpha of Gly734. A recombinant protein comprising the core of Pn (Ser1-Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with Pn activase a heterooligomeric Pn enzyme that has full catalytic activity (35 U/nmol). Treatment of the activated complexes with oxygen led to cleavage of the 14 kDa proteins into 11 and 3 kDa polypeptides as expected for the localization of the putative glycyl radical at Gly102 (YfiD) or Gly95 (Y06I). For the isolated fragments from Y06I, mass spectrometric analysis (nanoESI-MS) determined a C-terminal serine carboxamide in the 11 kDa fragment, and a N-terminal oxalyl modification in the 3 kDa fragment. We speculate that YfiD in E. coli and other facultative anaerobic bacteria has evolved as a "spare part" for Pn's glycyl radical domain, utilized for rapid recovery of PFL activity land thus ATP generation) in cells that have experienced oxidative stress.