Figure 2.
The midpoint potential of Mia40 is approximately −290 mV. Redox titration of the dithiol/disulfide couple of Mia40 was performed in a DTT or glutathione redox buffer. Equilibration was achieved at 2 h at pH 7.0 under anaerobic conditions. Data in all titrations were fit to the Nernst equation for a two-electron carrier. (A) Redox titrations were performed with mBBr. The best fit to the mBBr fluorescence magnitude versus Eh value was obtained with an Em of −277 mV (n = 3). (B) Redox titrations were performed as in A and intrinsic tryptophan fluorescence was used to monitor the redox state of Mia40. The best fit to the data were for a single n = 2 component with Em = −289 mV (n = 3). (C) Mia40 samples were incubated in aerobic conditions at the indicated redox (Eh) values. Samples were treated with AMS, which adds a molecular mass of 0.5 kDa. As controls, reduced (20 mM DTT) and reduced-denatured (95°C + 20 mM DTT) samples were included, demonstrating the addition of two and six equivalents of AMS, respectively (n = 3).