Figure 2. Demonstration of reverse and forward electron transport through complex I in isolated rat skeletal muscle mitochondria in cuvette-based assays assessed using the effect of rotenone challenge on NAD(P)H redox state; parallel superoxide/hydrogen peroxide production from sites I_Qr and I_Qf and its suppression by S1QELs.

(A–F) Cuvette-based assays of NAD(P)H autofluorescence (Ex 365/Em 450). In (A,B) 0% reduction was taken as the signal after 5 min with no substrate added; in (C–F), with 50 µM glutamate plus malate (G + M) added at the start, 0% reduction was taken as the average autofluorescence signal of all runs on the same day at 5 min in the absence of added 50 µM glutamate plus malate as in (A,B). One hundred percent reduction was taken as the steady value in each trace after the addition of 5 mM glutamate plus malate. (G,H) Cuvette-based assays of superoxide/hydrogen peroxide production measured as hydrogen peroxide using the Amplex UltraRed assay. (A,B,G) Reverse electron transport achieved by adding 12.5 mM glycerol 3-phosphate as sole substrate at 5 min and demonstrated in (B) by oxidation of the matrix NAD pool on challenge with 4 µM rotenone; (C–F,H) Forward electron transport achieved by adding 50 µM glutamate plus 50 µM malate in the reaction mix and 12.5 mM glycerol 3-phosphate as additional substrate at 5 min and demonstrated in (D–F) by reduction of the matrix NAD pool on challenge with 4 µM rotenone. (E–H) 1 µM S1QEL1.1 or S1QEL2.1 as indicated added of DMSO vehicle to the reaction mix; the same volume of DMSO was added to the relevant controls. Traces are representative of duplicates each day and at least three repeats on independent mitochondrial preparations.