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1.
FIGURE 9.

FIGURE 9. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

The effects of catalase (1000 units/ml) on the decay of preformed DEPMPO/HOO. The experiments used NADPH plus wtTrxR (A) or NADPH alone (B). Other experimental details were the same as those described in the legend to Fig. 8.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
2.
FIGURE 7.

FIGURE 7. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

Comparison of site-directed variants to wild-type TrxR. Representative ESR spectra of reactions incubated aerobically at 37 °C for 30 min. Each reaction contained 1.07 μm TrxR (wild type or the site-directed variants indicated), 14 mm DEPMPO, and 0.4 mm NADPH. The components of the spectra corresponding to DEPMPO/HO are indicated by black dots, whereas the components corresponding to DEPMPO/HOO are indicated by open diamonds. DEPMPO adducts were not seen when NADPH was omitted (not shown). Instrument settings were as described in the legend to Fig. 1.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
3.
FIGURE 10.

FIGURE 10. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

The relative rates of NADPH oxidation for the TrxR variants using H2O2 as the substrate. Both cuvettes contained Tris-EDTA buffer (pH 7.4), 0.4 mm NADPH, and 5 mm H2O2. The sample cuvette also contained TrxR (0.1 μm). The rates at 37 °C (determined by the change in absorbance at 340 nm, mean ± S.D. (error bars), n = 3) were taken from the linear portion during the first 50 s of the assay.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
4.
FIGURE 2.

FIGURE 2. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

Representative effects of SOD and MnTBAP on the DEPMPO adducts generated by TrxR under aerobic conditions. a and c, a 30-min incubation (37 °C) of 1.07 μm TrxR, 0.4 mm NADPH, and 14 mm DEPMPO. Spectra b and d are the same as a and c with the addition of SOD (333 units/ml) (b) or MnTBAP (0.2 mm) (d). e, ferrous ammonium sulfate plus H2O2 incubated for 10 min. f, the same as e with the addition of SOD. The instrument settings were the same as for Fig. 1.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
5.
FIGURE 6.

FIGURE 6. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

Representative effects of inhibitors of the Sec active site. Each spectrum was recorded after a 30-min aerobic incubation (37 °C) of 1.07 μm TrxR, 0.4 mm NADPH, and 14 mm DEPMPO. The samples for b and d were identical to those for a and c, respectively, except that each contained a TrxR inhibitor, either cisplatin (b) or 2,4-DNCB (d). The sample for e was the same as for d except that it also contained SOD (333 units/ml). The instrument settings were the same as for Fig. 1. In c and d, the components of the spectrum corresponding to DEPMPO/HO are indicated by black dots above the spectrum, whereas the components corresponding to DEPMPO/HOO are indicated by open diamonds.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
6.
FIGURE 4.

FIGURE 4. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

The effects of catalase, H2O2, ANF, metal chelators, and Fe(II) on the DEPMPO adducts generated by wtTrxR under aerobic conditions. A, 30-min incubations (37 °C) of 0.535 μm TrxR, 0.4 mm NADPH, and 14 mm DEPMPO with the following additions as indicated: catalase (833 units/ml), 50 μm H2O2, or 4 μm ANF. B, 30-min incubations (37 °C) of 1.07 μm TrxR, 0.4 mm NADPH, and 14 mm DEPMPO with the following additions as indicated: 0.2 mm DTPA, 0.2 mm DFX, or 15 μm Fe(II) as ferrous ammonium sulfate. The ESR instrument settings were the same as for Fig. 1. Both graphs show the relative molar amounts of DEPMPO/HO and DEPMPO/HOO (mean ± S.D. (error bars) for triplicate experiments) for the different treatments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus the wtTrxR samples.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
7.
FIGURE 11.

FIGURE 11. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

The generation of free HO by wtTrxR and the truncated and U498C variants in the presence (right) and absence (left) of catalase (833 units/ml). The samples were incubated aerobically for 30 min at 37 °C and included NADPH (0.4 mm), PBN (50 mm), DMSO (5%, v/v), and the indicated forms of TrxR (1.07 μm). The spectra spanned 3380–3580 G, but only the portion in which signals were observed is shown here. Computer simulations of the spectra at the left are consistent with a mix of PBN/OCH3 (hyperfine splitting constants aN = 15.05 G, aH = 3.32 G) and PBN/CH3 (hyperfine splitting constants aN = 16.51 G, aH = 3.68 G) adducts (see Fig. 3). The instrument settings were the same as described in the legend to Fig. 1, except that the microwave frequency was 9.806 GHz.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
8.
FIGURE 5.

FIGURE 5. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

Dependence of the DEPMPO adducts on O2. A, representative ESR spectra of reactions incubated aerobically (top) or anaerobically (bottom). The buffers, water, and NADPH were mixed and incubated for 5 min at 37 °C. Each reaction was started by adding TrxR (1.07 μm final concentration) and DEPMPO (14 mm final concentration). After 12 min at 37 °C, the reaction was loaded into an ESR flat cell and sealed, and spectra were acquired at room temperature. For the anaerobic experiments, the buffers and deionized water were preincubated in the anaerobic chamber for ≥24 h before use, and small volumes of NADPH, DEPMPO, and TrxR were pre-equilibrated for ≥4 h. The aerobic experiments were conducted identically, except all solutions and steps were handled under room air. Instrument settings were as described in the legend to Fig. 1. B, relative quantities of the HO, O, and carbon radical adducts of DEPMPO (mean ± S.D. for triplicate experiments) observed under aerobic versus anaerobic conditions. **, p < 0.01; ***, p < 0.001 versus the corresponding aerobic samples.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
9.
FIGURE 3.

FIGURE 3. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

A, representative ESR spectra in which PBN (50 mm) plus DMSO (5%) were substituted for DEPMPO. The samples contained NADPH (0.4 mm) and were incubated for 30 min at 37 °C. Spectrum a included TrxR (1.07 μm), whereas b was without TrxR. B, an expanded view of spectrum a is shown at the center, spanning the region from 3440 to 3500 G, and is compared with computer simulations of the PBN/OCH3 adduct spectrum (c) (hyperfine splitting constants aN = 15.05 G, aH = 3.32 G) and with a simulated PBN/CH3 adduct spectrum (d) (hyperfine splitting constants aN = 16.51 G, aH = 3.68 G). In spectrum a, the components of the signal corresponding to PBN/OCH3 are indicated by open dots, and those for PBN/CH3 are indicated by black dots. The PBN signals probably underestimate the amount of HO generated because O can mediate the decay of PBN/CH3 and PBN/OCH3 to ESR silent species (71), and the rate constant for the reaction of PBN with CH3 is about 3–4 orders of magnitude lower than the reaction of HO with DEPMPO or DMSO (72). The instrument settings were the same as described in the legend to Fig. 1.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
10.
FIGURE 8.

FIGURE 8. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

Representative data showing the relative amounts of DEPMPO/HOO (A, C, and E), and DEPMPO/HO (B, D, and F) for experiments in which preformed DEPMPO/HOO was the substrate. DEPMPO/HOO was preformed by incubating KO2 with DEPMPO as described under “Experimental Procedures.” After 1 min at room temperature, SOD (1000 units/ml) was added. One minute later, 0.4 mm NADPH and/or wtTrxR or one of the TrxR variants (1.07 μm) were added. The sample was loaded into the flat cell, and 30 consecutive ESR spectra were collected, with an acquisition time of 2.8 min per spectrum (84 min total). Controls lacking both NADPH and TrxR (none) were also analyzed. The time indicates the start time for the acquisition of each spectrum, with 0 min indicating the start of acquisition of the first spectrum. It takes ∼2 min to load the flat cell and tune the instrument, so 0 min is actually 2 min after adding the NADPH and/or TrxR. ESR instrument settings were as follows: modulation amplitude, 1 G; microwave power, 20.02 milliwatts; receiver gain, 6.32 × 104; time constant, 81.92 ms; microwave frequency, 9.77 GHz; sweep width, 200 G; field set, 3480 G; modulation frequency, 100 kHz; resolution, 2048 points/scan; scan time, 167.8 s; 30 consecutive scans (n = 1 each).

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
11.
FIGURE 12.

FIGURE 12. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

Scheme of the inherent NADPH oxidase activity of TrxR and source of the resulting ESR findings. Electrons from NADPH are transferred to the FAD, which reduces the N-terminal dithiol (Cys59/Cys64). This dithiol reduces the C-terminal Cys497/Sec498 active site. During normal catalysis, TrxR cycles between the 2- and 4-electron reduced states shared mainly between Cys59/Cys64 and Cys497/Sec498, with the FAD in the oxidized state or forming a charge-transfer complex with Cys64 (1, 7). In the absence of an electron-accepting substrate (e.g. oxidized thioredoxin), TrxR can reduce O2 to O. Mutants lacking Sec498 (Sec) generate less DEPMPO/HO but are still capable of generating significant O. However, mutants lacking Cys59 and/or Cys64 generate almost no O or HO signals, suggesting that this dithiol is a major site of O generation and that the FAD does not directly generate O. O is required for the majority of the DEPMPO/HO signal because SOD eliminates nearly all of this signal. With wtTrxR, the bulk of the signal is not the O adduct but rather the HO adduct, which is mostly formed by the 2-electron reduction of DEPMPO/HOO by TrxR; this process does not require H2O2 but does require the Sec (U498) in the reduced state (*) because the Cys59/Cys64 dithiol is also required to reduce the DEPMPO/HOO adduct. The 2-electron reduction of DEPMPO/HOO to DEPMPO/HO probably reflects its peroxidase activity because the 2-electron reduction of H2O2 similarly requires Sec in the reduced state. H2O2 can slow the rate of reduction of DEPMPO/HOO to DEPMPO/HO by acting as a competing substrate for the Sec-dependent peroxidase. A lesser amount of the DEPMPO/HO adduct can be attributed to the generation of HO in an H2O2-dependent but Sec-independent mechanism (Minor Pathway), as supported by the PBN/DMSO data (Figs. 3 and 11). The peroxidase activity of wtTrxR would be predicted to diminish the generation of free HO.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.
12.
FIGURE 1.

FIGURE 1. From: The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities.

Representative ESR spectra over time using a constant amount of TrxR and the spin trap DEPMPO. A, a fixed concentration of TrxR (1.07 μm) was incubated at 37 °C under room air for the times indicated with 0.4 mm NADPH and 14 mm DEPMPO. The spectrum at the bottom was also for 30 min but lacked NADPH. B, hydroxyl radical positive control consisting of H2O2 (1.7 mm), ferrous ammonium sulfate (0.15 mm), and DEPMPO (14 mm) (10-min incubation at 37 °C under room air). The components of the signal corresponding to DEPMPO/HO are indicated by black dots. C, superoxide positive control consisting of xanthine (2 mm), xanthine oxidase (0.2 units), DTPA (0.1 mm), DEPMPO (50 mm), and 50 mm potassium phosphate, pH 7.4 (3-min incubation at 37 °C under room air). The components of the signal corresponding to DEPMPO/HOO are indicated by open diamonds. The structures for the DEPMPO/HO and DEPMPO/HOO adducts are shown beside traces B and C, respectively. D, two different time courses (5–30 min and 10–120 min) showing the amounts of DEPMPO/HO and DEPMPO/HOO adducts over time in experiments with 1.07 μm TrxR plus NADPH (0.4 mm). E, a third time course (30–360 min) using the conditions as defined in D. F, relationship between the amount of TrxR and the amount of DEPMPO/HO adduct (r = 0.969) using a single incubation time of 30 min with different amounts of TrxR plus 0.4 mm NADPH and 14 mm DEPMPO. ESR instrument settings were as follows: modulation amplitude, 1 G; microwave power, 19.92 milliwatts; receiver gain, 6.32 × 104; time constant, 40.96 ms; microwave frequency, 9.76 GHz; sweep width, 200 G, field set, 3480 G; modulation frequency, 100 kHz; scan time, 42 s; number of scans, 9.

Qing Cheng, et al. J Biol Chem. 2010 July 9;285(28):21708-21723.

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