Ligand Access to the Active Site in Thermus thermophilusba3 and Bovine Heart aa3 Cytochrome Oxidases

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• 作者：William McDonald ; Chie Funatogawa ; Yang Li ; Istvan Szundi ; Ying Chen ; James A. Fee ; C. David Stout ; 脫l枚f Einarsd贸ttir
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：January 29, 2013
• 年：2013
• 卷：52
• 期：4
• 页码：640-652
• 全文大小：729K
• 年卷期：v.52,no.4(January 29, 2013)
• ISSN：1520-4995

文摘

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 NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic 鈥渨ay station鈥? 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.