The challenge for small-molecule probes of dithiol/disulfide-type oxidoreductases is to distinguish selectively between proteins that perform similar chemistries. The TrxR and GR systems that are the major mammalian dithiol/disulfide reductants (Fig. ) have been well reviewed. In brief: (i) TrxRs and GRs are low-expression enzymes (ca. 20 nM cellular concentration) that harvest electrons from NADPH to dithiol/disulfide active sites (CVNVGC motif). GR has high specificity for reducing glutathione disulfide (GSSG) and little activity on other species. The major TrxRs in mammals are cytosolic TrxR1 and mitochondrial TrxR2. They have a broader substrate scope than GR, due to their additional C-terminal selenolthiol/selenenylsulfide active site (CU motif) located on an exposed flexible tail, that relays electrons from the NADPH-driven dithiol site to substrates. TrxR's native substrates include disulfide Trxs and TRP14; diverse small molecules can also be reduced (Fig. ). The rare selenolthiol of TrxR endows it with distinctive redox properties compared to dithiol proteins, including enhanced reaction kinetics, lowered reduction potential, and a resistance to permanently function-suppressing oxidation. (ii) Trxs and dithiol Grxs are proteins with exposed dithiol/disulfide redoxactive sites (CxxC motif) that reduce a broad scope of substrates including disulfides; they have moderate expression levels (up to ca. 10 µM). Trxs have lower reduction potentials and are reduced by TrxR; Grxs have higher reduction potentials and use monothiol GSH (ca. 2-5 mM in the cytosol) for recovery via a net trimolecular reaction. Fig. | Mechanistic considerations towards designing TrxR-selective probes. a, Simplified view of the central dithiol/disulfide-type redox cascades of cell biology. b, Reaction mechanism for TrxR reducing a generalised dichalcogenide XY, using its C-terminal Sec-Cys active site. Initial exchange (1) gives an intermediate that can either evolve backwards by reforming the dichalcogenide (2) or else evolve forwards by full reduction (3) to the dichalcogenol. Recovery of TrxR by NADPH makes this a non-equilibrium process. c, Topology considerations: linear topology dichalcogenides are irreversibly committed to cargo release after monothiol exchange, whereas the exchange and reduction steps for cyclic topology dichalcogenides are reversible (full mechanism in Fig. ).