Prokaryotic and eukaryotic cytochrome c oxidases and several bacterial ubiquinol oxidases compose a superfamily of heme-copper oxidases. These enzymes are terminal components of aerobic respiratory chains, the principal energy-generating systems of aerobic organisms. Two such heme-copper oxidases are the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides and the bo-type ubiquinol oxidase of Escherichia coli. These enzymes catalyze the reduction of oxygen to water at a heme-copper binuclear center. Energy conservation is accomplished by coupling electron transfer through the metals of the oxidases to proton translocation across the cellular membrane. The Rb. sphaeroides and E. coli enzymes have previously been utilized in site-directed mutagenesis studies which identified two histidines which bind the low-spin heme (heme a), as well as additional histidine residues which are probable ligands for copper (Cub). However, the histidine that binds the heme of the binuclear center (heme a3) could not be unequivocally identified between two residues (His284 and His419). Additional characterization by Fourier transform infrared spectroscopy of the CO-bound forms of the E. coli enzyme in which His284 is replaced by glycine or leucine demonstrates that these mutations cause only subtle changes to CO bound to the heme of the binuclear center. Resonance Raman spectroscopy of the Rb. sphaeroides enzyme in which His284 is replaced by alanine shows that the iron-histidine stretching mode of heme a3 is maintained, in contrast with the loss of this mode in mutants at His419. These results demonstrate that His284 is not the heme a3 ligand. Therefore, the remaining conserved histidine within subunit I of the oxidases (His419) is proposed to be the heme a3 ligand. In this model, the axial ligands of the two hemes are located within a single helix and thus are connected by a pathway of covalent bonds. The implications of this model on the control of electron transfer through the enzyme are discussed.