Methaneseleninic acid is a substrate for truncated mammalian thioredoxin reductase: implications for the catalytic mechanism and redox signaling

Abstract

Mammalian thioredoxin reductase is a homodimeric pyridine nucleotide disulfide oxidoreductase that contains the rare amino acid selenocysteine (Sec) on a C-terminal extension. We previously have shown that a truncated version of mouse mitochondrial thioredoxin reductase missing this C-terminal tail will catalyze the reduction of a number of small molecules. Here we show that the truncated thioredoxin reductase will catalyze the reduction of methaneseleninic acid. This reduction is fast at pH 6.1 and is only 4-fold slower than that of the full-length enzyme containing Sec. This finding suggested to us that if the C-terminal Sec residue in the holoenzyme became oxidized to the seleninic acid form (Sec-SeO(2)(-)) that it would be quickly reduced back to an active state by enzymic thiols and further suggested to us that the enzyme would be very resistant to irreversible inactivation by oxidation. We tested this hypothesis by reducing the enzyme with NADPH and subjecting it to high concentrations of H(2)O(2) (up to 50 mM). The results show that the enzyme strongly resisted inactivation by 50 mM H(2)O(2). To determine the redox state of the C-terminal Sec residue, we attempted to inhibit the enzyme with dimedone. Dimedone alkylates protein sulfenic acid residues and presumably will alkylate selenenic acid (Sec-SeOH) residues as well. The enzyme was not inhibited by dimedone even when a 150-fold excess was added to the reaction mixture containing the enzyme and H(2)O(2). We also tested the ability of the truncated enzyme to resist inactivation by oxidation as well and found that it also was resistant to high concentrations of H(2)O(2). One assumption for the use of Sec in enzymes is that it is catalytically superior to the use of cysteine. We and others have previously suggested that there are reasons for the use of Sec in enzymes that are unrelated to the conversion of substrate to product. The data presented here support this assertion. The results also imply that the redox signaling function of the thioredoxin system can remain active under oxidative stress.

Figure 1

Two important ways in which Se is involved in the catalytic mechanism. Se is involved in accepting electrons from the N-terminal redox center (governed by rate constant k_ex – top panel) as well as donating electrons to the protein substrate Trx (governed by rate constant k_Nuc-Se – middle panel). The Sec residue of mammalian TR is part of the conserved Gly-Cys₁-Sec₂-Gly motif found on a flexible C-terminal extension of the enzyme. The N-terminal redox center (green) is on the opposite subunit of the homodimer. Reducing equivalents originate from NADPH and are passed onto the flavin, which in turn reduces the N-terminal disulfide. The top and middle panels only show two discrete steps in the catalytic cycle. For a complete description of the enzymatic mechanism of high M_r TRs, please see (28). Here, and previously, we have produced a truncated mTR missing the C-terminal redox center (bottom panel). This truncated mitochondrial mTR can reduce a number of small molecule substrates (SM) such as DTNB, lipoic acid, and selenite (10, 12). The use of the truncated mTR allows us to isolate and study the exchange step in greater detail. Note that Cys_IC denotes the interchange Cys residue that attacks either the selenosulfide bond of the C-terminal redox center or the small molecule substrate.

Figure 2

(A) Activity vs. pH profile for mTRΔ8. As can be seen, the truncated enzyme has a sharp pH optimum near 6 for the reduction of CH₃SeO₂⁻. (B) The N-terminal redox center has an acidic pH optimum for a number of different substrates: selenite (open diamonds), lipoic acid (closed circles), DTNB (open squares).

Figure 3

The importance of Se to substrate utilization by the N-terminal redox center. The N-terminal redox center will reduce small molecule Se-containing substrates such as SeO₃⁻², CH₃SeO₂⁻, and selenosulfide (Se–S) containing peptides. The S-analogs of these substrates are either not reduced at all such as HOCH₂SO₂⁻ (reported here) and SO₃⁻² (10), or are reduced very slowly as in the case of cystine and other disulfide (S–S) containing peptides (12). We believe that the most likely reason that these Se-containing compounds are good substrates (reduced quickly) compared to the S-analogs is due to selenium’s strong ability to accept electrons (high electrophilicity). The explanation that high electrophilicity is the determining factor in substrate utilization by the N-terminal redox center is consistent with the fact that DTNB, lipoic acid, and quinones, all highly electrophilic compounds, are turned over by the N-terminal redox center as reported by us and others ( and references therein).

Figure 4

Our truncated mTR construct (mTRΔ8) will reduce CH₃SeO₂⁻ as an external substrate (left structure). This implies (right structure) that if the Sec residue in the C-terminal tail, as part of the full-length enzyme, becomes oxidized to the Sec-SeO₂⁻ form, it can be easily reduced by either the N-terminal redox center or by the adjacent Cys residue (Cys₁).

Figure 5

(A) The plot monitors the consumption of NADPH by following the decrease in A₃₄₀. When 10 mM H₂O₂ is added to a reaction containing 50 nM mTR-GCUG and NADPH, mTR reduces H₂O₂ to water and becomes oxidized. The oxidized mTR is reduced by NADPH resulting in a decrease in A₃₄₀. After prolonged exposure (20 min) to excess H₂O₂, 90 μM Trx is added to the cuvette and the sharp increase in NADPH consumption (shown by a larger negative slope) shows that Trx is rapidly reduced and that a large excess of H₂O₂ does not inhibit the enzyme. (B) Comparison of activity progress curves for mTR treated with H₂O₂ (blue = 50 mM, green = 1 mM) and control mTR (red = no H₂O₂). After approximately 25 min, all of the NADPH in the reaction is consumed (for the enzyme treated with 50 mM H₂O₂) shown by a plateau in the slope. The samples were then treated with 14 units of catalase to remove excess H₂O₂ for 12 min. During this quenching step the A₃₄₀ was not monitored, but we have added a line to the plot for continuity. Since all of the NADPH was consumed in the sample treated with 50 mM H₂O₂, an additional bolus of NADPH was then added to the reaction to achieve a final concentration of 200 μM. The reaction was then monitored for 2 additional min at 340 nm to ensure all of the H₂O₂ was removed and then 90 μM Trx was added to each sample. The activity progress curves are extremely similar, even for the sample treated with 50 mM H₂O₂. The overall results show that mTR is very resistant to inactivation, even though the ability of the enzyme to turnover H₂O₂ shows that the Se-atom must be exposed to the oxidant.

Figure 6

Dimedone/WT mTR trapping experiment. In this experiment we are attempting to trap a selenenic acid (Enz-SeOH) intermediate by adding the sulfenic acid trapping reagent dimedone to the WT enzyme in the presence of H₂O₂. Here 10 nM mTR is added to a cuvette containing 50 mM H₂O₂ and 200 μM NADPH. As shown by the decrease in A₃₄₀, the enzyme is consuming NADPH due to the enzyme’s peroxidase activity. After 20 min we began a series of dimedone additions, starting with 500 nM dimedone and gradually increasing the concentration of dimedone to 150 μM. No change in the slope is apparent even after adding a large excess of dimedone to the reaction, showing the enzyme continues to turnover H₂O₂. After 40 min, a fresh bolus of NADPH is added and the peroxidase activity is monitored for several more minutes after which 90 μM Trx is added to the reaction. Upon addition of Trx, the consumption of NADPH is sharply increased as shown by the decrease in A₃₄₀.