Electron Paramagnetic Resonance Evidence for a Cysteine-Based Radical in Pyruvate Formate-lyase Inactivated with Mercaptopyruvate

Abstract

Pyruvate formate-lyase (PFL) is a glycyl radical-containing enzyme that catalyzes the reversible, nonoxidative conversion of pyruvate and CoA into acetyl-CoA and formate. The radical is located on the alpha-carbon of glycine 734 and is required for catalysis. Two cysteine residues, C418 and C419, are also essential for catalysis. Mercaptopyruvate, a biologically relevant pyruvate analog, is shown here to be a mechanism-based inactivator of PFL. Upon addition of mercaptopyruvate to active PFL, an EPR spectrum is generated which exhibits components from two sulfur-based radicals. For one of these radicals, a disulfide radical, the hyperfine coupling to a single beta-methylene hydrogen is resolved in features at g = 2.057 and 2.023. The effects of deuterium labeling of the enzyme on the EPR spectrum for this species are consistent with the new radical being on a cysteine residue, probably cysteine 418 or 419. This spectrum is not formed upon addition of the inactivator to mutant enzymes, C418S and C419S, indicating that both active site cysteines are required for formation of the new radicals. The identity of the second species is also ascribed to be a sulfur-based radical on the basis of the EPR feature found at g = 2.01. Our results constitute the first direct evidence of sulfur-based radical formation in an enzyme. A mechanism for formation of the cysteine-based disulfide radical is proposed which requires the participation of the two active site cysteines as well as the glycyl radical.

Bacteriophage T4 Anaerobic Ribonucleotide Reductase Contains a Stable Glycyl Radical at Position 580

“…In support of such a model are the facts that the E. coli anaerobic ribonucleotide reductase has been observed to be inhibited by class I and class II mechanism-based inhibitors (34) and that the k cat of 3 s Ϫ1 obtained in this study for the T4 anaerobic reductase closely matches the k cat of 4 -11 s Ϫ1 found for the E. coli class I enzyme (30,39,40). Recently, the pyruvate formatelyase enzyme has been identified to contain a thiyl radical at its active site (41). We are currently investigating the role of conserved cysteine residues as potential members of an anaerobic ribonucleotide reductase active site that would be homologous to the active site cysteines in the aerobic class I reductase.…”

High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5′-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase

“…The powder pattern is dominated by an axially asymmetric g anisotropy with principal values of g 1 ϭ 2.023, g 2 ϭ 2.015, and g 3 ϭ 2.002. These g values eliminate a thiyl radical (28)(29)(30) and a persulfide radical (28,36,37) as the source of this signal and are consistent with a disulfide radical anion (Table 1) (28,(38)(39)(40). Finally, we also note in the low-field edge of the spectrum a long, weak tail (Fig.…”

Cysteinyl and Substrate Radical Formation in Active Site Mutant E441Q of Escherichia coli Class I Ribonucleotide Reductase

“…In pyruvate formate lyase a disulfide radical is formed when the enzyme is inactivated with mercaptopyruvate, and a sulfinyl (and a peroxyl) radical is formed when pyruvate formate lyase is inactivated in the presence of oxygen (21,22). The disulfide radical has g x ϭ 2.057, g y ϭ 2.023, g z Ϸ 2.00, and the sulfinyl radical has g x ϭ 2.0204, g y ϭ 2.0084, g z ϭ 2.0005.…”