Site-directed Mutagenesis Reveals the Essentiality of the Conserved Residues in the Putative Diiron Active Site of the Trypanosome Alternative Oxidase

• Published in: The Journal of biological chemistry Vol. 277; no. 10; pp. 8187 - 8193
• Main Authors: Ajayi, Wilfred U., Chaudhuri, Minu, Hill, George C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.03.2002; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Motifs; Animals; Binding Sites; Cell Division; Escherichia coli - metabolism; Genetic Complementation Test; Glutamine - chemistry; Heme - chemistry; Histidine - chemistry; Immunoblotting; Iron - metabolism; Models, Genetic; Mutagenesis, Site-Directed; Mutation; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; Trypanosoma brucei; Trypanosoma brucei brucei - enzymology; trypanosome alternative oxidase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111477200

Trypanosoma brucei possesses a non-cytochrome, salicylhydroxamic acid (SHAM)-sensitive ubiquinol:oxygen oxidoreductase known as trypanosome alternative oxidase (TAO). TAO and similar SHAM-sensitive alternative oxidases (AOXs) contain 2–3 conserved diiron-binding motifs (EXXH). Site-directed mutagenesis of residues H165A, E214A, E266A, and H269L within the conserved EXXH motif abolished the ability of TAO to complement the heme-deficient Escherichia colistrain GE1387. These mutations also reduced the growth of this E. coli auxotroph to about 85% of the control cells containing wild type TAO. In contrast, mutation of residues outside the EXXH motifs, e.g. V205A, L243A, C261A, and V271A, had little effect on complementation, and the reduction in the cell growth was about 5–10%. Mutations of the putative iron-binding residues within the EXXH motifs of TAO abolished the ability to confer SHAM-sensitive respiration to E. coliheme mutant, whereas mutations of the non-conserved/non-iron binding residues resulted in 20–30% reduction of SHAM-sensitive respiration of the E. coli auxotroph. Immunoblot analysis of the total cellular protein of transformed E. coli revealed that the expression level of mutated and wild type TAO (35 kDa) remained unaltered. Mutation at C261A produced a truncated but functional protein of 28 kDa. The addition of ortho-phenanthroline to the growth medium produces a non-functional TAO. The effect ofortho-phenanthroline on the activity of TAO was completely alleviated by the addition of iron in the medium, which suggests that iron is needed for the activity of TAO. This work demonstrates that His-165, Glu-214, Glu-266, and His-269 and the presence of iron are essential for the activity of TAO.