Diversity of chemical mechanisms in thioredoxin catalysis revealed by single-molecule force spectroscopy

Abstract

Thioredoxins (Trxs) reduce disulfide bonds via a Michaelis-Menten mechanism. Upon substrate stretching at high forces, an SN2 reaction can be used by bacterial Trxs. A third mechanism, single-electron transfer, is now revealed in Trxs of either bacterial or eukaryotic origin, and is correlated with the depth of the Trx substrate-binding groove. Thioredoxins (Trxs) are oxidoreductase enzymes, present in all organisms, that catalyze the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single-molecule level. Here we use single-molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different Trx enzymes. All Trxs show a characteristic Michaelis-Menten mechanism that is detected when the disulfide bond is stretched at low forces, but at high forces, two different chemical behaviors distinguish bacterial-origin from eukaryotic-origin Trxs. Eukaryotic-origin Trxs reduce disulfide bonds through a single-electron transfer reaction (SET), whereas bacterial-origin Trxs show both nucleophilic substitution (SN2) and SET reactions. A computational analysis of Trx structures identifies the evolution of the binding groove as an important factor controlling the chemistry of Trx catalysis.