title: Single-molecule studies of disulfide bond reduction pathways used by human thioredoxin.
abstract: Disulfide bond reduction pathways used by human thioredoxin (hTrx) are studied at the single molecule level using a recombinant protein (I27SS)8. (I27SS)8 contains eight tandem repeats of identical immunoglobulin-like modules with one disulfide bond in each module. Single (I27SS)8 molecules are stretched at constant force applied by a cantilever in a force-clamp mode of atomic force microscopy (FC-AFM). Disulfide reduction events are accurately detected from stepwise increases in the end-to-end length of (I27SS)8. Earlier FC-AFM studies observed one disulfide reduction pathway used by hTrx and suggested an additional electron tunneling mechanism. Here, a very large set of unbiased FC-AFM data is collected in a range of clamping forces. By analyzing the data using exponential fits and dwell time histograms two disulfide reduction pathways used by hTrx are resolved. Based on previous studies one of these pathways is attributed to force-dependent Michaelis-Menten catalysis. The latter reduction pathway is weakly force-inhibited and occurs sporadically. Bimolecular nucleophilic substitutions (SN2) and electron tunneling (ET) mechanisms are discussed to explain the second pathway. Direct SN2 and ET mechanisms cannot be discounted, but a hypothetical E2-SN2 mechanism involving a hydride reducing a disulfide bond provides an interesting alternative, which needs to be verified in future experiments.
authors: Department of Physics, Kansas State University, 307 Cardwell Hall, Manhattan, KS, USA. szosz@ksu.edu
authors: Robert
authors: R
authors: Szoszkiewicz
mesh_terms: D055162:Biocatalysis; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018625:Microscopy, Atomic Force; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D013879:Thioredoxins