Fig. 3. Small-molecule ligands bias specificity of plant POR (SbPOR2b) to reduce diverse electron acceptors.

A Effects of small-molecule ligands on SbPOR2b activity in proteoliposomes using Cytc and RS as electron acceptors. B Dose-response curves of rifampicin, cyclophosphamide and dhurrin in the Cytc and RS assays, respectively. Rifampicin acts as an agonist towards Cytc enhancing its reduction rate and inverse agonist towards RS reducing the reduction rate. Cyclophosphamide displays the reverse effect acting as an inverse agonist towards Cytc reduction and agonist towards RS reduction. Dhurrin acts as an agonist towards both Cytc and RS reduction at low micromolar concentrations. The fact that ligands display differential effects on SbPOR2b activity to reduce the two electron acceptors indicates biased specificity of POR. IC50 values are extracted from the Hill equation. A, B The bar plots and dose-response curves represent the mean ± SD of independent replicates normalized to controls with propagated error (n = 3–12; see Supplementary Fig. 9 for raw data and Supplementary Table 3 for exact value of n for each experimental condition). All data are corrected for potential ligand photophysical effect (see Supplementary Fig. 7). Note, overlapping data points appear shaded. Level of significance is determined by one-way ANOVA and Tukey’s HSD test correcting for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.005; see Supplementary Material for details). Source data are provided as a Source Data file.