Peroxide modification of monoalkylated glutathione reductase. Stabilization of an active-site cysteine-sulfenic acid.

Hydrogen peroxide reacts with two-electron reduced glutathione reductase (GR EH2 species) to give the native oxidized enzyme (E) without detectable intermediates. Prior alkylation of the EH2 interchange thiol with iodoacetamide, however, dramatically changes both the course and overall rate of the peroxide reaction. This oxidation, monitored spectrally, is characterized by an intermediate (EHRint) with enhanced long wavelength absorbance extending to 800 nm. This species decays in a second peroxide-dependent phase to an enzyme form (EHRox) easily distinguished from E. Quenching experiments with catalase allow the isolation of a stable mixture consisting of 36% monoalkylated GR (EHR), 60% EHRint, and 4% EHRox; NADPH titration and anaerobic dithiothreitol addition lead to quantitative reduction of EHRint to EHR, and there is an increase in thiol titer of 0.8-SH/FAD on NADPH reduction. Of the four titratable thiols present in EHR, 2.7 are lost on oxidation to EHRox and 0.7-0.8 mol of cysteic acid/FAD is formed. On the basis of these and other observations, we conclude that alkylation of the EH2 interchange thiol, which blocks disulfide formation, allows peroxide reaction at the remaining charge-transfer thiol to proceed via a stabilized cysteine-sulfenic acid intermediate (EHRint), which undergoes further oxidation to the corresponding cysteic acid (EHRox).