Inactivation of pyruvate formate-lyase by dioxygen: defining the mechanistic interplay of glycine 734 and cysteine 419 by rapid freeze-quench EPR.

Pyruvate formate-lyase from Escherichia coli (EC 2.3.1.54; PFL) catalyzes the reversible anaerobic conversion of pyruvate and CoA into acetyl-CoA and formate. Active PFL contains a novel alpha-carbon centered glycyl radical at G734 that is required for its catalytic activity. Two adjacent cysteine residues, C418 and C419, are essential for PFL activity according to site-directed mutagenesis studies. Upon exposure to air, active PFL loses its activity with the concomitant loss of the glycyl radical. Previous EPR studies of dioxygen inactivation of PFL revealed protein-based peroxyl and sulfinyl radicals during the manual mixing and quenching process [Reddy et al. (1998) Biochemistry 37, 558-563]. To probe the mechanism of this process, we carried out experiments using rapid freeze-quench EPR spectroscopy. Upon mixing of active wild type or C418A PFL with oxygenated solution, a short-lived radical intermediate appears at the earliest time point (10 ms), followed by the appearance of a long-lived sulfinyl radical. The axial EPR spectrum of this short-lived radical (g = 2.034, 2.007) is characteristic of a peroxyl radical. When C419A PFL or the double mutant [C418A/C419A] PFL was mixed with oxygenated solution, the peroxyl radical was also observed at 10 ms but in this case persisted over 12 s. These observations provide compelling evidence to support a proposed mechanism in which dioxygen quenches the glycyl radical in the active enzyme and the resulting peroxyl radical may react further with the sulfhydryl group of the C419 residue to form the sulfinyl radical.