Ligand Access to the Active Site in <i>Thermus thermophilus</i> <i>ba</i>₃ and Bovine Heart <i>aa</i>₃ Cytochrome Oxidases

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O₂ carriers, we recently found that NO and O₂ binding in <i>Thermus thermophilus</i> (<i>Tt </i>) <i>ba</i>₃ is ∼10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallography, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild-type <i>Tt ba</i>₃ and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O₂ channel of <i>Tt ba</i>₃ are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the <i>aa</i>₃ enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type <i>ba</i>₃ and the T231F mutant. The results show that the <i>Tt ba</i>₃ Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic “way station”, which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in <i>ba</i>₃ and bovine <i>aa</i>₃ show conformational freedom of the bovine F238 and the F231 side chain of the <i>Tt ba</i>₃ Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme