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...
moreKnowledge 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 crystallog-
raphy, 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 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 ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.
