Cryo-EM Structures Reveal the Unique Binding Modes of Metyltetraprole in Yeast and Porcine Cytochrome <i>bc</i>₁ Complex Enabling Rational Design of Inhibitors

Cytochrome <i>bc</i>₁ (complex III) represents a significant target for the discovery of both drugs and fungicides. Metyltetraprole (MET) is commonly classified as a quinone site inhibitor (Q_oI) that combats the G143A mutated isolate, which confers high resistance to strobilurin fungicides such as pyraclostrobin (PYR). The binding mode and antiresistance mechanism of MET remain unclear. Here, we determined the high-resolution structures of inhibitor-bound <i>S. cerevisiae</i> complex III (MET, 2.52 Å; PYR, 2.42 Å) and inhibitor-bound porcine complex III (MET, 2.53 Å; PYR, 2,37 Å) by cryo-electron microscopy. The distinct binding modes of MET and PYR were observed for the first time. Notably, the MET exhibited different binding modes in the two species. In <i>S. cerevisiae</i>, the binding site of MET was the same as PYR, serving as a <i>Pm</i>-type inhibitor of the Q_o site. However, in porcine, MET acted as a dual-target inhibitor of both Q_o and Q_i. Based on the structural insights, a novel inhibitor (YF23694) was discovered and demonstrated excellent fungicidal activity against downy mildew and powdery mildew fungi. This work provides a new starting point for the design of the next generation of inhibitors to overcome the resistance.