S1QELs suppress mitochondrial superoxide/hydrogen peroxide production from site I_Q without inhibiting reverse...

Mitochondria are important sources of superoxide and hydrogen peroxide in cell signaling and disease. In particular, superoxide/hydrogen peroxide production during reverse electron transport from ubiquinol to NAD⁺ though Complex I is implicated in several physiological and pathological processes. S1QELs are small molecules that suppress superoxide/hydrogen peroxide production at Complex I without affecting forward electron transport. Their mechanism of action is disputed. To test different mechanistic models, we compared the effects of two inhibitors of Complex I electron transport (piericidin A and rotenone) and two S1QELs from different chemical families on superoxide/hydrogen peroxide production and electron transport by Complex I in isolated mitochondria. Piericidin A and rotenone (and S1QEL1.1 at higher concentrations) prevented superoxide/hydrogen peroxide production from sites I_Q and I_F in Complex I by inhibiting reverse electron transport into the complex. S1QELs decreased the potency of electron transport inhibition by piericidin A and rotenone, suggesting that S1QELs bind directly to Complex I. S1QEL2.1 (and S1QEL1.1 at lower concentrations) suppressed site I_Q without affecting reverse electron transport or site I_F, showing that sites I_Q and I_F are distinct, and that S1QELs do not work simply by decreasing reverse electron transport to site I_F (or site I_Q). S1QELs did not affect the reduction of NAD⁺ or the rate of site I_F driven by reverse electron transport, therefore they do not alter the driving forces for reverse electron transport and that is not how they suppress site I_Q. We conclude that S1QELs bind to Complex I to influence the conformation of the piericidin A and rotenone binding sites and directly suppress superoxide/hydrogen peroxide production at site I_Q, which is a separate site from site I_F.