Ligand access to the active site in Thermus thermophilus ba(3) and bovine heart aa(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 Thermus thermophilus (Tt ) ba3 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 Tt ba3 and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O2 channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa3 enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba3 and the T231F mutant. The results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede N...