Stepwise reduction of cytochromesb-562,b-566 andb-558 in rat liver mitochondria

1. Addition of KCN to aerobic, rotenone-inhibited rat liver mitochondria without addition of substrate caused reduction of cytochromesb-562(having an α-band at 562 nm at room temperature),c + c 1 anda + a 3. The effect of KCN on cytochromeb-562 was reversed by pentachlorophenol, though the effect of KCN on cytochromesc+c 1 anda+a 3 was not reversed by this uncoupler. 2. Addition of ATP to aerobic, rat liver mitochondria inhibited with 500 μM KCN under conditions where cytochromesb-562,c+c 1 anda+a 3 were reduced, caused reduction of cytochromeb-566. The absorbance spectrum of cytochromeb-566 had an α-band at 565.5 nm, a β-band at 538 nm and a γ-band at 431 nm, but no shoulder around 558 nm at room temperature. 3. Addition of succinate to rotenone-KCN-inhibited and ATP-treated rat liver mitochondria under conditions where cytochromesb-566,b-562,c+c 1 anda+a 3 were already fully reduced, caused reduction of cytochromeb-558 (having an α-band at 558 nm, a β-band at 527 nm and a γ-band at 426 nm at room temperature) after exhaustion of molecular oxygen in the reaction medium, without any contribution from a long-wavelength species (cytochromeb-566). 4. It was concluded that the 558-nm band is not a short-wavelength shoulder of cytochromeb-566, but is due to a different species from cytochromeb-566.