Investigating OcwA Interactions with Electron Shuttles

For all of the NMR experiments, OcwA prepared in 20 mM potassium phosphate buffer (pH 7.6) with 100 mM KCl was lyophilized and resuspended in D₂O. An excess of sodium dithionite was used to reduce the protein. ¹H NMR experiments were performed on a Bruker Avance II 500 MHz spectrometer equipped with a QXI probe for ¹H detection and an SEX probe for ³¹P detection. All NMR data were processed in the TopSpin 3.2 software. ¹H NMR spectra were acquired before and after lyophilization to ensure that protein integrity was preserved.
To study the influence of the electron shuttles on OcwA, NMR experiments were performed as previously described (31 (link)) using antraquinone-2,6-disulfonate (AQDS), flavin mononucleotide (FMN), riboflavin (RF), and phenazine methosulfate (PMS). Stock solutions of the different electron shuttles were prepared in 20 mM potassium phosphate buffer (pH 7.6) with 100 mM KCl. ¹H NMR spectra performed at 25°C on a Bruker Avance II 500 MHz NMR spectrometer equipped with a TCI cryoprobe for ¹H detection were acquired before and after the addition of the electron shuttles (molar ratios 0.5:1, 1:1, and 3:1 of electron shuttle to protein).
For the ³¹P-NMR binding experiments, samples containing 100 μM FMN prepared in 20 mM phosphate buffer (pH 7.6) with 100 mM KCl were titrated against increasing concentrations of OcwA at 25°C.