Studies on cytochrome b-555 from larvae of the housefly, Musca domestica L. I. Purification and properties of cytochrome b-555.

Abstract

1. Cytochrome b-555 was highly purified from larvae of the housefly, Musca domestica L. The absorption spectra of the purified cytochrome b-555 showed peaks at 358–360, 414, and 530mμ in the oxidized form, and at 424, 528, and 555mμ in the reduced. The α-band of the reduced form was asymmetric at room temperature and at neutral pH, but split into two distinct peaks at 552 and 556 mμ at liquid nitrogen temperature. The purified cytochrome contained protoheme as the prosthetic group and combined neither with carbon monoxide nor with cyanide. 2. In the ultracentrifugation, the cytochrome preparation showed a symmetrical pattern and its sedimentation coefficient, S20, w, was 1.43S. The molecular weight was determined to be 13, 700 by the gel filtration method. 3. Cytochrome b-555 was reduced readily by sodium dithionite, slowly by cysteine, and anaerobically by ascorbate, but was not reduced non-enzymatically by NADH. The reduced form was oxidized by potassium ferricyanide, beef heart ferricytochrome c, and very slowly by air (the apparent first-order velocity constant was 0.0046 sec−1). The midpoint redox potential (E'o) of cytochrome b-555 was +0.006volt at pH7.0 and at 12°C. 4. Purified cytochrome b-555 was reduced by NADH in the presence of the larval microsomal fraction or NADH-cytochrome b5 reductase [EC 1.6.2.2] purified from rat liver microsomea. The reaction was inhibited neither by antimycin A nor by rotenone as in the case of microsomal NADH-cytochrome b5 reductase systems of housefly larvae and mammalian liver. 5. Purified cytochrome b-555 seemed to be a solubilized form of cytochrome b5 which existed both in the mitochondrial and microsomal fractions prepared from the larvae. Solubilization of larval cytochrome b5 from the cytoplasmic membranes was discussed.