THE FREE-RADICAL IN PYRUVATE FORMATE-LYASE IS LOCATED ON GLYCINE-734

Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively C-13-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central C-13 nucleus (A parallel-to = 4.9 mT and A perpendicular-to = 0.1 mT) and to C-13 nuclei in alpha and beta-positions agree with literature data for glycine radical models. N-coupling was verified through uniform N-15-labeling. The large H-1 hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha-proton, which in the enzyme radical is readily solvent-exchangeable. Oxygen destruction of the radical produced two unique fragments (82 and 3 kl)a) of the constituent polypeptide chain. The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.