Abstract
Oxidation rates of the 2 cytochromes c and the 3 cytochromes b in mitochondria from the hypocotyls of etiolated mung beans (Phaseolus aureus) have been measured with a rapid mixing flow apparatus coupled to a dual wavelength spectrophotometer. The mitochondria were depleted with uncoupler and ADP prior to starting the experiments to minimize the complications caused by energy-linked processes on electron transport. The half-time for oxidation and absorbance change at a given wavelength for the rapidly oxidized cytochromes was plotted as a function of wavelength, with 570 mmu as reference, to yield a spectrum in which absorbance maxima and half-times were correlated. The 2 cytochrome c components have oxidation half-times of 2 milliseconds for c(549) and 3 milliseconds for c(547). (The subscripts refer to the difference absorbance maxima observed for these cytochromes in reduced-minus-oxidized difference spectra obtained at -196 degrees . The maxima observed at room temperature are 550 mmu for c(547) and 552 mmu for c(549).) Cytochrome b(557) (room temperature maximum 560 mmu) was oxidized with a half-time of 8 milliseconds, while b(553) (room temperature maximum 556 mmu) was oxidized relatively slowly with a half-time of 0.5 sec. Cytochrome b(562) (room temperature maximum 565 mmu) was oxidized with half-times in the range 15 to 35 milliseconds, depending on mitochondrial preparation. It was shown that b(562) is only partially reduced by succinate in anaerobic mitochondria previously depleted with uncoupler and ADP; this cytochrome can be fully reduced by succinate in coupled mitochondria under conditions where endogenous pyridine nucleotide is also reduced. Both b(553) and b(557) in depleted mitochondria are reduced in anaerobiosis by succinate. With ascorbate plus N,N'-tetramethylphenylenediamine as substrate, b(553) is only partially reduced in anaerobiosis, while b(557) is completely reduced in depleted mitochondria. This observed difference in ease of reducibility provides another method for differentiating the 3 cytochromes b of mitochondria isolated from plant tissues.