Srx catalytic mechanism. Srx catalyzes the reduction of the sulfinate function of PrxSO₂ via a phosphotransferase and a reductase step leading to a covalent thiosulfinate intermediate with Srx. The subsequent recycling of the Srx activity involves an external reductant R. P, Prx; S, Srx; C_P, peroxidatic Cys; C_R, regeneration Cys; C₈₄, Srx catalytic Cys; R, thiol reductant.

Kinetics of the reaction catalyzed by Srx monitored by P_i release. A, reaction of 10 μm wild-type Srx with 100 μm C171A PrxSO₂ in the presence of 1 mm ATP/MgCl₂ without added reductant (black trace) or in the presence of 50 mm DTT (light gray trace) or 235 μm Trx (dark gray trace) was monitored by the purine nucleoside phosphorylase-coupled assay (see text). Reactions were carried out in buffer TK at 30 °C. The progress curves are corrected from blank traces collected in the absence of Srx. Kinetics without reductant are described by an exponential process with a rate constant of 2.2 ± 0.4 min⁻¹ and an amplitude corresponding to 9 μm P_i released. In the presence of Trx or DTT as reductant, initial rate constants of 1.7 ± 0.1 and 2.2 ± 0.4 min⁻¹ are measured, respectively. B, comparison of the kinetics of P_i release for the reaction of C48A/C106A Srx (10 μm) with 100 μm C171A PrxSO₂ in the presence of 1 mm ATP/MgCl₂ without reductant (black trace) or in the presence 50 mm DTT (light gray trace) or 235 μm Trx (dark gray trace). Kinetics without reductant are described by an exponential process with a rate constant of 1.5 ± 0.3 min⁻¹ and an amplitude corresponding to 11 μm P_i released. The kinetics with Trx are described by an exponential process with a rate constant of 1.5 ± 0.7 min⁻¹ and an amplitude corresponding 13 μm P_i released, followed by a slower steady-state linear phase with rate constants of 0.25 ± 0.07 min⁻¹. In the presence of DTT as reductant, an initial rate constant of 1.2 ± 0.2 min⁻¹ is measured.

Nonreducing SDS-PAGE analysis of the species formed during the Srx-catalyzed reaction in the absence or presence of ATP. Equimolar concentrations of C171A PrxSO₂ and wild type (lanes 1 and 2), C106A (lanes 3 and 4), and C48S (lanes 5 and 6) Srxs (30 μm) were mixed in the absence (lanes 1, 3, and 5) or presence of 1 mm ATP-MgCl₂ for 10 min (lanes 2, 4, and 6), immediately followed by addition of 25 mm iodoacetamide and nonreducing SDS-PAGE fractionation. Reactions were carried out in buffer TK at 30 °C. The additional band migrating between the Prx monomer and Prx·Srx complex in lane 4 corresponds to a dimer of unspecifically oxidized Srx C106A.

Kinetics of formation of Cys⁴⁸/Cys⁸⁴-oxidized Srx species. The reaction of 100 μm wild-type PrxSO₂ with 10 μm wild-type Srx in the presence of 1 mm ATP and MgCl₂ was followed at 30 °C by analysis of aliquots of the reaction mixture quenched by acidification in 0.1% trifluoroacetic acid by reverse phase chromatography and monitored by absorption spectrophotometry at 215 nm. A, chromatogram of the reaction mixture for the retention times corresponding to the Srx species after 15 s incubation (solid line) and after 1 min incubation (dashed line). B, kinetics of the evolution of the area of the peak corresponding to the Cys⁴⁸/Cys⁸⁴ S. cerevisiae Srx species eluted at 14.58 min (■) analyzed as a monoexponential process (solid line) with a rate constant of 1.8 ± 0.3 min⁻¹.

Kinetics of reduction of the Cys⁴⁸/Cys⁸⁴Srx disulfide bond by E. coli Trx. The Trx fluorescence quenching associated with disulfide bond formation was recorded on a stopped-flow apparatus at 30 °C in buffer TK by mixing reduced Trx and Cys⁴⁸/Cys⁸⁴-oxidized C106A Srx (10 μm). Excitation wavelength was set at 295 nm, and emitted light was collected using a 320 nm cutoff filter. Data collected at various concentrations of reduced Trx were fitted to Equation 1. The deduced k_obs values plotted versus Trx concentration (■) were then fitted against Equation 2, which gave k_{obs, max} and K_S values of 105 ± 7 s⁻¹ and 210 ± 30 μm (solid line). Inset, typical time course obtained for 250 μm Trx.

Kinetics of reaction of S. cerevisiae Trx1 C33S with Srx activated by 2-thiopyridine. The reaction of 10 μm SrxC84(2-thiopyridine) (A) and SrxC48 (2-thiopyridine) (B) with variable concentrations of S. cerevisiae Trx1 C33S was monitored on a rapid kinetics spectrophotometer by the release of pyridine 2-thiolate at 343 nm. Reactions were carried out in buffer TK at 30 °C. Kinetic data (not shown) were fitted against Equation 1. The deduced k_obs values were plotted versus Trx concentration (●). A, data were fitted against Equation 2, which gave k_{obs, max} and K_S constants of 3.3 s⁻¹ and 151 μm (solid line). B, data were fitted to a linear relationship (solid line) with a slope corresponding to a k₂ value of 1.16·10⁵ m⁻¹·s⁻¹.

Proposed catalytic mechanism of Srx from S. cerevisiae. The recycling mechanism involves the attack of the Cys⁴⁸ of Srx on the sulfur atom of Cys⁸⁴ of the thiosulfinate intermediate, leading to the release of the oxidized Cys⁴⁸/Cys⁸⁴ Srx with intramolecular disulfide bond and of the sulfenic acid PrxSOH product. The Cys⁴⁸/Cys⁸⁴ Srx is then reduced by attack of the Trx catalytic Cys on Cys⁴⁸ to form a Trx-Srx mixed disulfide, followed by regeneration of reduced Srx by attack of the recycling Cys of Trx. P, Prx; S, Srx; C_P, peroxidatic Cys; C₈₄, Srx catalytic Cys; C₄₈, Srx regeneration Cys; T, thioredoxin.