title: Rheostat re-wired: alternative hypotheses for the control of thioredoxin reduction potentials.
abstract: Thioredoxins are small soluble proteins that contain a redox-active disulfide (CXXC). These disulfides are tuned to oxidizing or reducing potentials depending on the function of the thioredoxin within the cell. The mechanism by which the potential is tuned has been controversial, with two main hypotheses: first, that redox potential (Em) is specifically governed by a molecular 'rheostat'-the XX amino acids, which influence the Cys pKa values, and thereby, Em; and second, the overall thermodynamics of protein folding stability regulates the potential. Here, we use protein film voltammetry (PFV) to measure the pH dependence of the redox potentials of a series of wild-type and mutant archaeal Trxs, PFV and glutathionine-equilibrium to corroborate the measured potentials, the fluorescence probe BADAN to measure pKa values, guanidinium-based denaturation to measure protein unfolding, and X-ray crystallography to provide a structural basis for our functional analyses. We find that when these archaeal thioredoxins are probed directly using PFV, both the high and low potential thioredoxins display consistent 2H+:2e- coupling over a physiological pH range, in conflict with the conventional 'rheostat' model. Instead, folding measurements reveals an excellent correlation to reduction potentials, supporting the second hypothesis and revealing the molecular mechanism of reduction potential control in the ubiquitous Trx family.
authors: Department of Chemistry, Boston University, Boston, Massachusetts, United States of America.
authors: Kathryn D
authors: KD
authors: Bewley
authors: Howard Hughes Medical Institute, Cambridge, Massachusetts, United States of America; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors: Mishtu
authors: M
authors: Dey
authors: Howard Hughes Medical Institute, Cambridge, Massachusetts, United States of America; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors: Rebekah E
authors: RE
authors: Bjork
authors: Department of Chemistry, Boston University, Boston, Massachusetts, United States of America.
authors: Sangha
authors: S
authors: Mitra
authors: Department of Chemistry, Boston University, Boston, Massachusetts, United States of America.
authors: Sarah E
authors: SE
authors: Chobot
authors: Howard Hughes Medical Institute, Cambridge, Massachusetts, United States of America; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors: Catherine L
authors: CL
authors: Drennan
authors: Department of Chemistry, Boston University, Boston, Massachusetts, United States of America.
authors: Sean J
authors: SJ
authors: Elliott
mesh_terms: D001105:Archaea / Q000737:chemistry; D018360:Crystallography, X-Ray; D005978:Glutathione / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017510:Protein Folding; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D013879:Thioredoxins; D005978:Glutathione