Tetramethyl p phenylenediamine tmpd

This method was adapted from previously published protocols^{19 (link),57 (link)}. All measurements were performed using an OROBOROS Oxygraph-2k (Oroboros Instruments, Innsbruck, Austria). For measurement of total cellular respiration, intact cells (2 × 10⁶ cells/ml) were used and maintained in full growth medium supplemented with 20 mM HEPES during measurement. For direct measurement of mitochondrial respiration, digitonin-permeabilised cells (2 × 10⁶ cells/ml) were used and maintained in mitochondrial respiration buffer MiR05 during measurement. To induce respiration, 10 mM glutamate, 5 mM malate, 1 mM ADP, and 5 µg/ml digitonin were added. The following complex-specific ETC inducers were used: For complex II, 10 mM succinate (from Sigma, #S3674); for complex III, 1 mM tetramethylhydroquinone / duroquinol (from TCI Germany, Eschborn, Germany; #T0822); for complex IV, 50 µM tetramethyl-p-phenylenediamine (TMPD; from Sigma, #87890) supplemented with 200 µM ascorbate.