Pathways of electron flow established by tetramethylphenylenediamine in mitochondria and ascites tumor cells.

Abstract

Summary 1. The pathways of electron flow established by N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) have been studied in rat-liver mitochondria and ascites tumor cells. 2. Ascorbate-TMPD oxidation results in reduction of cytochrome c and a in the absence or presence of antimycin A. The reaction gives ADP:O ratios in excess of unity but these are lowered by antimycin A or high TMPD concentrations, conditions that eliminate oxidation of endogenous substrates. 3. Ascorbate-TMPD reversed electron flow and oxidation, occurring simultaneously, cause reduction of pyridine nucleotide, flavoprotein, and cytochromes in the aerobic steady state. However, when antimycin A is present, cytochrome b and other carriers on the substrate side of the inhibition site are oxidized, and components on the O2 side remain in the reduced state. Therefore, in the presence of ascorbate, TMPD is presumed to interact with the respiratory chain at cytochrome c (or c1 which has not been measured). 4. A TMPD bypass causes an oxidation of cytochrome b in antimycin-treated mitochondria. This bypass permits electron transfer over the inhibited site and results in ADP:O ratios of about 1 and 2 respectively with succinate and β-hydroxy-butyrate, reflecting a loss of energy conservation in the cytochrome b region. Despite loss of phosphorylation, ADP (and uncouplers) can reduce cytochrome b in the ascor-bate-TMPD, but not in the TMPD system. TMPD also restores the antimycin-inhibited respiration of ascites tumor cells. The bypass stimulates cell respiration and abolishes the apparent Crabtree phenomenon seen in the presence of mannose or oligomycin. 5. Mechanisms are considered whereby TMPD and ascorbate-TMPD affect reactions of cytochrome b through oxidation, reversed electron flow, and bypass pathways of electron flow.