Stable selones in glutathione-peroxidase-like catalytic cycle of selenonicotinamide derivative

Abstract

Selenonicotinamide, 2,2′-diselenobis[3-amidopyridine] (NictSeSeNict) exhibits glutathione-peroxidase (GPx)-like activity, catalyzing the reduction of hydrogen peroxide (H₂O₂) by glutathione (GSH). Estimated reactivity parameters for the reaction of selenium species, according to the Dalziel kinetic model, towards GSH (ϕ_GSH) and H₂O₂ (ϕ_H2O2), indicated that the rate constant for the reaction of NictSeSeNict with GSH is higher as compared to that with H₂O₂, indicating that the activity is initiated by reduction. ⁷⁷Se NMR spectroscopy, HPLC analysis, mass spectrometry (MS) and absorption spectroscopy were employed to understand the nature of selenium intermediates responsible for the activity. The ⁷⁷Se NMR resonance at 525 ppm due to NictSeSeNict disappeared in the presence of GSH with the initial appearance of signals at δ 364 and 600 ppm, assigned to selone (NictCSe) and selenenyl sulfide (NictSeSG), respectively. Reaction of H₂O₂ with NictSeSeNict produced a mixture of selenenic acid (NictSeOH) and seleninic acid (NictSeO₂H) with ⁷⁷Se NMR resonances appearing at 1069 and 1165 ppm, respectively. Addition of three equivalents of GSH to this mixture produced a characteristic ⁷⁷Se NMR signal of NictSeSG. HPLC analysis of the product formed by the reaction of NictSeSeNict with GSH confirmed the formation of NictCSe absorbing at 375 nm. Stopped-flow kinetic studies with global analysis revealed a bimolecular rate constant of 4.8 ± 0.5 × 10³ M⁻¹ s⁻¹ and 1.7 ± 0.6 × 10² M⁻¹ s⁻¹ for the formation of NictCSe produced in two consecutive reactions of NictSeSeNict and NictSeSG with GSH, respectively. Similarly the rate constant for the reaction of NictCSe with H₂O₂ was estimated to be 18 ± 1.8 M⁻¹ s⁻¹. These studies clearly indicated that the GPx activity of NictSeSeNict is initiated by reduction to form NictSeSG and a stable selone, which is responsible for its efficient GPx activity.