Kinetic studies of the reactions of O₂ and NO with reduced Thermus thermophilus ba₃ and bovine aa₃ using photolabile carriers

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• 作者：Ó ; lö ; f Einarsdó ; ttir^a ; ^{olof@ucsc.edu} ; Chie Funatogawa^a ; Tewfik Soulimane^b ; ^c ; Istvan Szundi^a
• 关键词：Thermus thermophilus ba3 ; O2 and NO photolabile carriers ; Double-laser transient absorption spectroscopy ; CO photodissociation
• 刊名：Biochimica et Biophysica Acta (BBA)/Bioenergetics
• 出版年：2012
• 期刊代码：13_00052728
• 类别：bio
• 出版时间：April, 2012
• 卷：1817
• 期：4
• 页码：672-679
• 文件大小：820 K

摘要

The reactions of molecular oxygen (O₂) and nitric oxide (NO) with reduced Thermus thermophilus (Tt) ba₃ and bovine heart aa₃ were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. To determine whether the photodissociated carbon monoxide (CO) in the CO flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence of CO using photolabile O₂ and NO complexes. The binding of O₂/NO to reduced ba₃ in the absence of CO occurs with a second-order rate constant of 1 脳 10⁹ M^鈭?#xA0;1 s^鈭?#xA0;1. This rate is 10-times faster than for the mammalian enzyme, and which is attributed to structural differences in the ligand channels of the two enzymes. Moreover, the O₂/NO binding in ba₃ is 10-times slower in the presence of the photodissociated CO while the rates are the same for the bovine enzyme. This indicates that the photodissociated CO directly or indirectly impedes O₂ and NO access to the active site in Tt ba₃, and that traditional CO flow-flash experiments do not accurately reflect the O₂ and NO binding kinetics in ba₃. We suggest that in ba₃ the binding of O₂ (NO) to heme a₃^2 + causes rapid dissociation of CO from Cu_B⁺ through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to Cu_B⁺. These findings indicate that structural differences between Tt ba₃ and the bovine aa₃ enzyme are tightly linked to mechanistic differences in the functions of these enzymes. This article is part of a Special Issue entitled: Respiratory Oxidases.