Interplay of proton and electron transfer to determine concerted behavior in the proton-coupled electron transfer of glutathione oxidation.

Glutathione (GSH), whose thiol group dictates its redox chemistry, is oxidized to the thiyl radical (GS˙), which rapidly dimerizes to GSSG. Previously, we found that the oxidation rate of GSH by IrCl62- depends on the base (B) concentration and the pKa of its conjugate acid BH+, so that collateral to a stepwise mechanism, the concerted pathway GSH + IrCl62- + B = GS˙ + IrCl63- + BH+ was proposed as the rate determining step. Herein, this investigation is extended to include oxidant-base pairs that render exothermic and endothermic conditions of ΔG°' for electron transfer (ET) and proton transfer (PT). Experiments were conducted by the reaction of GSH with an electrogenerated oxidant M+ and using digital simulations to infer the mechanism. Data analysis shows that despite parallel mechanisms, the concerted one seems to predominate for the oxidant-base pair that renders the most isoenergetic coupled state, whereby a PT with is capable of producing an ET with , as a result of the Nernstian shift of with pKa. In contrast, the stepwise PT-ET appears to dominate when GS- grows in stability as becomes more negative. Understanding the interplay between ET and PT will help in the design of catalysts for energy harvesting processes that rely on proton-coupled electron transfer.

Probing the Ca2+/CaM-induced secondary structural and conformational changes in calcineurin.

Calcineurin (CN) is a Ca(2+)/CaM-dependent Ser/Thr protein phosphatase that plays a critical role in coupling Ca(2+) signals to a cellular response. Various methods have been applied to explore CN activation. A widely accepted model involves CaM binding to the CaM-binding domain (CN 389-413), inducing displacement of the CN autoinhibitory peptide (CN 467-486) from the active site. However, almost the entire regulatory region (CN 374-521), except the autoinhibitory peptide, is not visible in the electron density map of the reported structures. In the present study, we determined the overall secondary structure of CN in the presence or absence of Ca(2+)/CaM using FT-IR, and the Ca(2+)/CaM-induced structural dynamics and conformational changes were monitored by hydrogen-deuterium exchange experiments. The results revealed that the regulatory domain possessed some intrinsic structure. The binding of Ca(2+) and subsequent binding of CaM generated a sequential folding of CN, transforming it into a more constrained, less flexible conformation.