| Although the cytochrome bo terminal oxidase in Vitreoscilla is similar in many respects to the E. coli enzyme, it differs significantly in that it functions as a primary Na + pump in contrast to the H+ pumping cytochrome bo in E. coli. The operon encoding the terminal oxidase for ubiquinol oxidation in Vitreoscilla cyo was isolated, cloned, and sequenced. It contains five genes which encode subunits I, II, III, and IV of the oxidase and heme farnesyl transferase. The operon contains a typical Shine-Dalgarno sequence, an E. coli-like promoter, and several possible binding sites for regulatory proteins. The sequence conservation of the Vitreoscilla cytochrome bo subunits was shown by their greater than 50% amino acid sequence similarities with corresponding proteins from other bacteria. In addition, hydropathy plots of all five cyo proteins showed strong similarity with those of the corresponding E. coli subunits. It has been shown that Vitreoscilla cytochrome bo can function as a Na+ pump instead of a H+ pump. Whether subunit I, the largest subunit, could be involved in sodium pumping is an open question. In order to investigate this suggestion, the entire cyo operon, including the natural cyo promoter, was cloned into vector pHG165, and transformed into E. coli GV100, a partial deletion mutant of cytochrome bo. This system allowed measurement of both Na+ pumping and cytochrome bo function for both wild type Vitreoscilla cytochrome bo and (site-directed) mutants in the protein's subunit I. Four subunit I amino acids (Five mutants) that are conserved and may be crucial to Vitreoscilla cytochrome bo function, were altered using site-directed mutagenesis, and tested for NADH stimulated oxidase activity, ubiquinol oxidase activity, and Na+ pumping ability using this system. Amino acids, M115R, M115K, K361Q, K361M, and E285Q, were found to be crucial for these two functions. In this context the potential roles of these residues in enzyme function are discussed. |
