Low-temperature spectral properties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata

Abstract

Highly purified mitochondrial preparations from the trypanosomatid hemoflagellate, Crithidia fasciculata (A.T.C.C. No.11745), were examined by low-temperature difference spectroscopy. The cytochrome a+ a 3 α maximum of hypotonically-treated mitochondria reduced with succinate, was shifted from 605 nm at room temperature to 601 nm at 77 °K. The Soret maximum, found at 445 nm at 23 °C, was split at 77 °K into two approximately equally absorbing species with maxima at 438 and 444 nm. A prominent shoulder observed at 590 nm with hypotonically-treated mitochondria was not present in spectra of isotonic controls. The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent. Unlike other soluble c-type cytochromes, the α maximum of cytochrome c 555 was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane. Using cytochrome c 555-depleted mitochondria, a cytochrome c 1 pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c 555-depleted preparations exhibited an α maximum at 553 nm at room temperature. The reduced rate of succinate oxidation by cytochrome c 555-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c 1, even in the presence of antimycin, indicated that cytochrome c 555-mediated electron transfer between cytochromes c 1 and a+ a 3 in a manner analagous to that of cytochrome c in mammalian mitochondria.