PREPARATION AND CHARACTERIZATION OF THE HYDROPHILIC CU-A CYTOCHROME-C DOMAIN OF SUBUNIT-II OF CYTOCHROME-C-OXIDASE FROM THERMOPHILIC BACILLUS-PS3

Cytochrome c oxidase of the thermophilic bacterium, PS3, was treated with trypsin. The hydrophilic domain of 26 kDa can be easily cleaved off from the hydrophobic anchor domain at the N-terminal region of subunit II, but remains attached to the rest of the enzyme upon gel-filtration in the presence of 0.2% lauroyl sarcosinate. The separation occurred in the presence of 5 M urea in addition to 0.2% lauroyl sarcosinate. After relatively prolonged proteolysis, that induced severe activity decay, and subunit I fragmentation, the 26 kDa fragment of subunit II can be easily isolated from the rest, suggesting that this fragment with cytochrome c and Cu-A interacts with subunit I. The separated fragment showed absorption spectra due to Cu-A and cytochrome c. Reconstitution of the cytochrome oxidase activity occurred on addition of the 26 kDa fragment to the proper gel-filtration chromatographic fraction.