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.

A Voltammetric Perspective of Multi-Electron and Proton Transfer in Protein Redox Chemistry: Insights From Computational Analysis of Escherichia coli HypD Fourier Transformed Alternating Current Voltammetry

“…As expected, the , and “best fit” n 1 = n 2 = 1 model parameter values for the pH 6.0 dataset are consistent with those from our previous work ( Adamson et al, 2017b ). Furthermore, the range covered by and (∼250 mV, from −95.2 to −348 mV) is consistent with previously observed mid-point potentials of disulfide bonds (∼300 mV) ( Chivers et al, 1997 ; Chobot et al, 2007 ; Bewley et al, 2015 ).…”

“…In this study, although H2ase-MFHypD serves as an extremely useful test system for developing our data analysis techniques for probing disulfide mechanisms, it is not possible to conclude if this redox-driven bond making/breaking is relevant to the physiological function of the enzyme ( Adamson et al, 2017b ; Nutschan et al, 2019 ). However, there are a multitude of proteins and enzymes where the disulfide chemistry is vitally important and in vitro electrochemical studies do provide a useful insight into the in vivo biological chemistry, as demonstrated by the comprehensive study by Bewley et al ( Bewley et al, 2015 ).…”

Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions

“…Mutational studies indicated that the cis-proline loop is also involved in stabilization of EcDsbA mixed disulfide intermediates with substrates in vivo [184], metal binding by the active site Cys residues [185]; and binding and positioning of the disulfide present in the substrate [93,186]. Studies with archeal Trxs are in an apparent contrast with the rheostat model [187], reinforcing the idea that there are multiple factors governing the reduction potentials of thiol-disulfide oxidoreductases, besides the composition of the amino acids in the C-X-X-C motif.…”

The role of sulfenic acids in cellular redox signaling: Reconciling chemical kinetics and molecular detection strategies

“…[9] The kinetics by which Cys-SH is oxidized is highly variable and is not simply dictated by the p K a of the involved Cys residue. [10–12] Second-order rate constants of H 2 O 2 -dependent Cys-SH oxidation range dramatically from 10 −3 to 10 8 M −1 s −1 , implying that adjacent or surrounding amino acids or other factors are capable of extensively tuning the reactivity of protein Cys-SH. [9] Similarly, the chemical properties of protein Cys-SOH species are diverse, including both nucleophilic as well as electrophilic properties, and are likely responsible for highly variable stability of this intermediate within diverse proteins.…”