Fig. 3.
Blocking movement of loop TMH1-2ND3 reversibly disengages the proton pumps. Proton pumping was monitored as 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quench in proteoliposomes containing either parental or mutant complex I treated with 5 mM dithiothreitol (DTT) (a, b) or 0.1 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) (c–f) for 5 min before starting the assay. As indicated, 0.5 µM ACMA, 70 µM decylubiquinone (DBQ). 100 µM NADH and 5 µM carbonylcyanide m-chlorophenylhydrazine (CCCP) were added during the assay. Addition of 5 mM DTT to DTNB treated proteoliposomes during the assay had no effect on parental complex I (e), but rapidly restored proton pumping of the mutant enzyme (f). Representative traces of one out of four datasets obtained with independent batches of proteoliposomes are shown