S1QELs suppress mitochondrial superoxide/hydrogen peroxide production from site IQ without inhibiting reverse electron flow through Complex I

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.