Dioxygen inactivation of pyruvate formate-lyase: EPR evidence for the formation of protein-based sulfinyl and peroxyl radicals

Sreelatha G. Reddy, Kenny K. Wong, Camran V. Parast, Jack Peisach, Richard S. Magliozzo, John W. Kozarich

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

We here report EPR studies that provide evidence for radical intermediates generated from the glycyl radical of activated pyruvate formate-lyase (PFL) during the process of oxygen-dependent enzyme inactivation, radical quenching, and protein fragmentation. Upon exposure of active PFL to air, a long-lived radical intermediate was generated, which exhibits an EPR spectrum assigned to a sulfinyl radical (RSO*). The EPR spectrum of a sulfinyl radical was also generated from the activated C418A mutant of PFL, indicating that Cys 418 is not the site of sulfinyl radical formation. Exposure of the activated C419A mutant or C418AC419A double mutant to air on the other hand, resulted in a new EPR spectrum that we assign to the α-carbon peroxyl radical (ROO*) of the active-site glycine, G734. These findings suggest that C419 is the site of sulfinyl formation and that replacement of this cysteine with alanine results in the accumulation of the carbon peroxyl radical. The results also support the proposal that the peroxyl radical and the sulfinyl radical are intermediates in the oxygen- dependent inactivation and cleavage of the protein. Moreover, these observations are consistent with the hypothesis that C419 and G734 are in close proximity in the activated enzyme and may participate in a glycyl/thiyl radical equilibrium. A mechanism that accounts for the formation of the radical intermediates is proposed.

Original languageEnglish
Pages (from-to)558-563
Number of pages6
JournalBiochemistry
Volume37
Issue number2
DOIs
StatePublished - 13 Jan 1998

Fingerprint

Dive into the research topics of 'Dioxygen inactivation of pyruvate formate-lyase: EPR evidence for the formation of protein-based sulfinyl and peroxyl radicals'. Together they form a unique fingerprint.

Cite this