Purification and properties of a diheme cytochrome b561 of the Escherichia coli respiratory chain.

H Murakami,K Kita,Y Anraku

doi:10.1016/s0021-9258(17)36126-4

Abstract

A new b-type cytochrome, cytochrome b561 (Murakami, H., Kita, K., Oya, H., and Anraku, Y. (1984) Mol. Gen. Genet. 196, 1-5) was purified to near homogeneity from the cytochrome b561-amplified Escherichia coli K12 strain HM204/pAM5029. The purified cytochrome b561 was a single polypeptide with a molecular weight of 18,000, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point was determined to be 9.6. The difference spectrum of the cytochrome at 77 K shows a major alpha-absorption peak at 561 nm and a minor peak at 555 nm. The absolute spectrum at room temperature of the oxidized form of the cytochrome had an absorption peak at 414 nm, and that of the reduced form had peaks at 562, 530, and 428 nm. The oxidation-reduction potential of the cytochrome was estimated to be +20 mV. The cytochrome contained 91.2 nmol of heme/mg of protein, showing that it was a cytoplasmic membrane-bound, b-type diheme cytochrome.

Highlights

Solubilization and Purification-Plasmid pAM5029 is the neity from the cytochrome bsel-amplifiedEscherichia most stable of the hybrid plasmids carrying cybB, the struccoli K12 strain HM2041pAM5029
77 K showsa major a- were solubilized with Triton X-100
The novel b-type cytochrome purified in thiswork fromthe inner cytoplasmic membrane of E. coli K12 contained two protohemes/18,000-dalton polypeptide and had a split a-absorption peak at 561 and 555 nm at 77 K

Summary

RESULTS

Ami, H., Kita, K., Oya, H., and Anraku,Y. (1984)Mol. Gen. Electrophoretic Studies-Purified cytochrome b56, was found to be almost homogeneous as judged by SDS-polyacrylamide gel electrophoresis (Fig. lA). Cytochrome b562-0 and b 5 ~ d as standard proteins Cyto- molecular weight of the cytochrome was 18,000, the pure chrome b556 is a low-potential cytochrome that transfers elec- cytochrome should contain 55.6 nmol of heme/mg of protein. The cytochrome b561 is present tion was 82.1%: in a wild type strain [7] and is reduced with respiratory Fig. 2 shows results on isoelectric focusing of cytochrome substrates [6],suggesting that itfunctions as acomponent of b561.

MATERIALS AND METHODS’

DISCUSSION

MATERIALS AND METHODS