Descriptive Analysis of Transient-State Observations for Thioredoxin/Glutathione Reductase (Sec597Cys) from Schistosoma mansoni

Abstract

Thioredoxin/glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both oxidized thioredoxin and glutathione with electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH). SmTGR is a drug target for the treatment of Schistosomiasis, an infection caused by Schistosoma platyhelminths residing in the blood vessels of the host. Schistosoma spp. are reliant on TGR enzymes as they lack catalase and so use reduced thioredoxin and glutathione to regenerate peroxiredoxins consumed in the detoxification of reactive oxygen species. SmTGR is a flavin adenine dinucleotide (FAD)-dependent enzyme, and we have used the flavin as a spectrophotometric reporter to observe the movement of electrons within the enzyme. The data show that NADPH fractionally reduces the active site flavin with an observed rate constant estimated in this study to be ∼3000 s −1 . The flavin then reoxidizes by passing electrons at a similar rate to the proximal Cys159−Cys154 disulfide pair. The dissociation of NADP + occurs with a rate of ∼180 s −1 , which induces the deprotonation of Cys159, and this coincides with the accumulation of an intense FAD-thiolate charge transfer band. It is proposed that the electrons then pass to the Cys596−Cys597 disulfide pair of the associated subunit in the dimer with a net rate constant of ∼2 s −1 . (Note: Cys597 is Sec597 in wild-type (WT) SmTGR.) From this position, the electrons can be passed to oxidized thioredoxin or further into the protein to reduce the Cys28− Cys31 disulfide pair of the originating subunit of the dimer. From the Cys28−Cys31 center, electrons can then pass to oxidized glutathione that has a binding site directly adjacent.

Assigning function to active site residues of Schistosoma mansoni thioredoxin/glutathione reductase from analysis of transient state reductive half-reactions with variant forms of the enzyme

“…Where possible, concentrations were determined spectrophotometrically using the following extinction coefficients: NADPH; ε 340 nm = 6,220 M −1 cm −1 , NADP + ; ε 260 nm = 17,800 M −1 cm −1 , thioredoxin (SmTrx); ε 280 nm = 9,500 M −1 cm −1 . The extinction coefficient of each SmTGR variant was determined by methods previously reported ( Smith et al, 2023 ): SmTGR U597C; ε 463 nm = 11,700 M −1 cm −1 , SmTGR C159S; ε 445 nm = 12,600 M −1 cm −1 , SmTGR U597S; ε 463 nm = 11,500 M −1 cm −1 , SmTGR C31S; ε 463 nm = 11,300 M −1 cm −1 , SmTGR H571A; ε 460 nm = 11,300 M −1 cm −1 , SmTGR Y296A; ε 455 nm = 11,500 M −1 cm −1 . All reactant concentrations indicated in this text are post-mixing.…”

“…The method of expressing and purifying all SmTGR variants were adapted from the previously established protocols utilized to isolate the U597C SmTGR variant ( Smith et al, 2023 ). The variants of interest include Cys159Ser (C159S), Cys597Ser (U597S), Cys31Ser (C31S), His571Ala (H571A), and Tyr296Ala (Y296A).…”

“…Prior to experimentation, the enzyme was thawed at room temperature and small amounts of precipitated enzyme were removed by centrifugation at 10,000 g for 10 min, and 30 kDa centrifugal filters were used to buffer exchange the supernatant enzyme into the desired reaction buffer (typically 50 mM KPi, pH 7.4). Additionally, a heat treatment (heating at 55°C for 5 min) was performed with variants that were sufficiently stable, to induce precipitation of apo enzyme ( Smith et al, 2023 ). Two relatively less stable variants, U597S and Y296A, did not undergo this additional step.…”

“…All SmTGR variants were quantified using the measured extinction coefficient of the FAD cofactor bound to the enzyme. Each variant’s extinction coefficient was determined according to published protocols ( Smith et al, 2023 ). Approximately 10 µL of a concentrated enzyme sample was diluted into 50 mM NaPi, pH 7.4 and an absorption spectrum recorded.…”

“…Recently, a transient-state characterization of Sm TGR U597C reductive half-reaction was completed ( Smith et al, 2023 ). Utilizing the spectrophotometric properties of the FAD and charge transfer intermediate species, a more direct depiction of the electron displacement within SmTGR was demonstrated.…”