HIGH-YIELD PURIFICATION OF A 4-SUBUNIT CAA(3)-TYPE CYTOCHROME-OXIDASEFROM THE THERMOPHILIC BACTERIUM BACILLUS PS3 USING FAST PROTEIN LIQUID-CHROMATOGRAPHY

The thermophilic bacterium, Bacillus PS3, was grown in a vigorously ae rated nutrient broth at 65 degrees C with 100 mM glutamic acid serving as a supplemental carbon and nitrogen source, These growth conditions resulted in membranes highly enriched in cytochrome c oxidase (COX) [ 23.32 +/- 4.32 nmol heme a/g of cells (n = 5)], which is nearly a thre efold higher concentration of COX (heme caa(3)-type) than previously r eported for this organism, A new high-yield purification of COX was pe rformed by extracting the bacterial membranes with Triton X-100 (7 mg/ mg protein), followed by ion-exchange fast liquid protein chromatograp hy using a QAE (trimethyl ammonium) resin with subsequent hydroxyapati te chromatography and ammonium sulfate fractionation. This purificatio n regime resulted in a 16% yield of cytochrome c oxidase with 20 mg of pure caa(3)-type COX (13 nmol heme a/mg protein) isolated from 100 g of cells, SDS-PAGE showed that the isolated enzyme had four subunits w ith apparent M(r) of 68, 38, 23, and 13 kDa, In addition, a new 34-kDa peptide was also detected in this preparation, which may represent th e ORF1 gene product for this organism, Subunit II (M, = 38 M) of the i solated enzyme was shown to contain covalently bound heme c by using b oth heme-staining of SDS-PAGE and immunoreactivity with an anti-cytoch rome c antibody, The purified enzyme also exhibited high electron tran sfer activity (340 s(-1)) when assayed at pH 6.5 in the presence of th e nonionic detergent, beta-dodecyl maltoside. (C) 1995 Academic Press, Inc.