Exploration of the molecular structure of Escherichia coli cytochrome bo ubiquinol oxidase by genetic approach

Cytochrome bo ubiquinol oxidase is one of the two terminal ubiquinol oxidases in the aerobic respiratory chain of Escherichia coli. By deleting the intergenic region between the cyoA and cyoB and one base in the overlapping sequence between cyoB and cyoC, in-frame fusions are made between all three subunits (II-I-III), the resulting gene product still assembles as part of a functional oxidase. The fused subunit (II-I-III) contains 22 transmembrane spans. These data support the previously proposed topology of the subunits. The purified cytochrome bo oxidase contains four subunits. The observed molecular weight of subunit II by mass spectroscopy is considerably less than the calculated value from the deduced amino acid sequence of its corresponding gene cyoA. The similarity of the N-terminal signal sequence of subunit II with those of known lipoproteins suggest that it is modified by lipids. This is proved by demonstrating that subunit II incorporates radioactive palmitic acid.A series of partial deletion mutants on subunit II have been constructed. Removal of the redundant C-terminal tail has no influence on the structure and function of the oxidase, whereas other deletion mutants are totally non-functional. Both the low-spin heme and the high-spin heme sites are severely disturbed, as manifested by the misincorporation of heme B into the binuclear center and the abnormal $\alpha$-peak in the reduced minus oxidized difference optical spectra. Some deletion mutants have not shown any ubiquinol oxidase activity. It is likely that subunit II is involved in quinol binding. Site-directed mutagenesis has been conducted on all the highly conserved residues in subunit II and certain residues that are conserved in the quinol oxidase but not in cytochrome c oxidase. Replacement of the charged Glu89 with neutral residues gives rise to non-functional mutant oxidases. Of the total 33 site-directed mutants constructed in this work, only the mutant W136A shows an unusually high K$\rm\sb{M}$ value when compared to the wild-type. The data suggest that Trp136 is involved in the quinol binding.Cytochrome bo oxidase is able to incorporate heme B and heme O. Comparison of oxidases purified from various strains with different proportions of heme B and heme O has led to the conclusion that the $bo\sb3$-type oxidase is much more active than the $oo\sb3$-type oxidase, and the $bb\sb3$-type is the least active form. Finally, it has been found that deletion of the genes for subunits III and IV leads to a weak interaction between subunit I and subunit II. When cyoC (subunit III) or cyoD (subunit IV) is deleted, the mutant oxidases inserted into the cytoplasmic membrane have no detectable ubiquinol-1 oxidase activity. It seems that subunit III and subunit IV are required for the assembly of a functional cytochrome bo oxidase.U of I OnlyETDs are only available to UIUC Users without author permissio