The protozoal parasite Trypanosoma brucei depends on a mitochondrial non-cytochrome terminal oxidase known as the trypanosome alternative oxidase (TAO) in its mammalian host. We have recently cloned the cDNA from T. brucei bloodstream form and have characterized a 33 kDa mitochondrial protein as TAO. Here we report that the TAO is a single copy gene in T. brucei and its expression is down regulated at the level of transcript abundance during differentiation from the bloodstream to the procyclic trypanosomes. Like other alternative oxidases (AOXs) cloned from different plants and fungi, TAO possesses the conserved sequences at the centrally located predicted membrane spanning domains and the signature sequence at the C-terminal hydrophilic domain for a pair of putative iron binding motifs (E-X-X-H). Phylogenetic analysis of the deduced protein sequences of eight different alternative oxidases cloned from different plants and fungi revealed that TAO is more closely related to the alternative oxidases of the fungi clade than that of plants. TAO has been functionally expressed in Escherichia coli. In the first of the two putative iron binding motifs, site-directed mutagenesis of E215 to A, L, N and Q resulted in the loss of the ability of the TAO gene to complement the heme deficiency of the E. coli mutants (SASX41B and GE1387) by conferring on them a CN-insensitive pathway of respiration. The conservative substitution of E215 by aspartate and histidine reduced the growth of the E. coli auxotrophs by approximately 80%. The mutations apparently did not have any effect on the stability of the expressed protein as revealed by the immunoblot analysis of the bacterial protein using TAO monoclonal antibody, which we have developed. Together, these points suggest that E215 plays an important role in the function of TAO. The steady state level of TAO mRNA is down-regulated in the procyclic stage presumably accounting for the low levels of TAO protein in these forms.