The reduction rate at a given force is obtained by summing, averaging and fitting to a single exponential numerous traces (15–80) like the one shown in Fig. 2b. The solid lines are fitting to the kinetic model. The grey circles and dashed lines represent the rate vs. force dependence for modern Trxs: (c) Pea Trxm from chloroplast, (d) P. falciparum Trx, (a,e) E. coli Trx and (f) Human Trx (all extracted from ref.16). These modern Trxs are descendants of the ancestral Trxs in the same plot. The error bars are given by the s.e.m. obtained using the bootstrap method.

(a) A high activity for AECA (squares) and LACA (circles) Trxs can be observed at pH 5 when the substrate is pulled at low forces (50–150 pN). LBCA Trx (triangles) shows similar activity than that at pH 7.2 also with a similar trend (Fig. 3a). The solid lines are exponential fit to the experimental data. (b) Rate constants for disulfide reduction by ancestral Trx enzymes are considerably higher than those measured for modern E. coli and human Trx. While thioredoxin from the acidophile Acetobater aceti shows activity at pH 5, enzymes from the thermophilic Sulfolobus tokodaii do not show a detectable rate of reduction at the same pH. All experiments were conducted at a pulling force of 100 pN. The error bars are given by the s.e.m. obtained using the bootstrap method. (c) Activity of ancestral Trxs and modern E. coli Trx measured using DTND as substrate at pH 5 and determined by monitoring spectrophotometrically the formation of TNB at 412 nm. The error bars represent s.d. from three different measurements.

(a) Schematic representation of the singe-molecule disulfide reduction assay. A first pulse of force rapidly unfolds the I27_G32C–A75C domains (11 nm step). When the disulfide bond is exposed to the solvent a single Trx molecule can reduce it (14 nm step) (b) Experimental force-clamp trace showing single disulfide reductions of a (I27_G32C–A75C)₈ polyprotein. The unfolding pulse was set at 185 pN for 0.2 s and the test-pulse force at 500 pN. (c) Probability of reduction (P_red(t)) resulted from summing and normalizing the reduction test pulse at different forces for AECA Trx (3.5 μM). (d) Force-dependency of disulfide reduction by AECA Trx; human Trx is also shown for comparison. Both Trx enzymes show a similar pattern: a negative force-dependency of the reduction rate, from 30–200 pN, consistent with a Michalis-Menten mechanisms and a force-independent mechanism, from 200 pN and up, described by an electron transfer reaction16. Notice the higher activity for AECA Trx (3.5 μM for AECA Trx vs. 10 μM for human TRX). The lines represent fittings to the kinetic model (see methods). The error bars are given by the s.e.m. obtained using the bootstrap method.

(a) Schematic phylogenetic tree showing the geological time in which different extinct organisms lived, i.e., last bacterial common ancestor (LBCA); last archaeal common ancestor (LACA); archaea/eukaryota common ancestor (AECA) and last eukaryotic common ancestor (LECA). Other internal nodes are: the last common ancestor of cyanobacterial and deinococcus/thermus groups (LPBCA), the last common ancestor of γ-proteobacteria (LGPCA), and the last common ancestor of animals and fungi (LAFCA). The dashed lines represent further bifurcations. Divergence times are compiled from multiple sources and are summarized in the Timetree of Life17. The figure indicated the global environment; although both aerobic and anaerobic organisms are found in modern environments. (b) Posterior probability distribution of the inferred amino acids across 106 sites for the interested internal nodes. The inferred amino acid at each site for the interested internal node is the residue with the highest posterior probability. (c) Denaturation temperatures (T_m) vs. geological time for ancestral Trx enzymes. Modern E. coli and Human Trx enzymes are also indicated. The dashed line represents a lineal fit to the data. The inset shows experimental DSC thermograms for E. coli Trx and LBCA Trx. The instrumental uncertainty of DSC measurements is < 0.5 °C.