Abstract
Two thioredoxins have been described in Escherichia coli, TrxA and Trx2. Both thioredoxins are capable of reducing disulfide bonds using a conserved pair of cysteine residues present in a WCGPC motif. A number of unique structural and regulatory features distinguish the Trx2 subfamily from the much larger TrxA family. The Trx2 subfamily has an additional N-terminal domain of +/- 30 residues, which contains two additional conserved CXXC motifs. Moreover, the gene coding for Trx2 is under control of the oxidative stress transcription factor OxyR in E. coli. This suggests that Trx2 may play a role in the cellular defense against oxidative stress. We show here that Trx2 contains zinc in a 1:1 stoichiometry, making it the first identified zinc-binding thioredoxin. The zinc atom is coordinated by the four cysteines of the two N-terminal CXXC motifs. The zinc center of Trx2 binds zinc with a very high affinity (Ka of >1018 m-1). We show that in vitro oxidation of the zinc binding cysteines by H2O2 releases the zinc and induces a conformational change. The zinc-free protein conserves its reductase activity. Altogether, our results suggest that the zinc center might play the role of a redox switch, changing a yet to be identified activity.
Highlights
Thioredoxins are small redox proteins present in many eukaryotic and prokaryotic cells
Sequence Analysis—One of the most striking differences between TrxA and Trx2 is the presence of an N-terminal domain containing two additional CXXC motifs arranged in the sequence CTHCX16CGRC
Thioredoxins have been shown to be responsible for thiol-disulfide exchange reactions and serve as electron donors for PAPS reductase, ribonucleotide reductase, and methionine sulfoxide reductase [1,2,3]
Summary
Cloning—E. coli genomic DNA was amplified with Pfu polymerase (Invitrogen). The PCR-amplified product was purified and cloned into a pET11a plasmid. To prepare zinc-saturated Trx with both catalytic cysteines oxidized (Trx2catcysox), the protein was incubated with a 10-fold excess of GSSG for 10 min at 4 °C and gel filtrated as explained above. The zinc-free oxidized protein was reduced with 2 mM DTT and loaded onto a second NAP-5 column equilibrated with the same buffer. Both buffer and DTT solution had been depleted from their metals by incubation with Chelex resin. After this treatment, Trx was reduced and contained less than 5% zinc. All reagents were depleted of their metals by incubation with Chelex resin (Bio-Rad)
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