Abstract
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 1×109M−1s−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 a32+ causes rapid dissociation of CO from CuB+ through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to CuB+. 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.
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More From: Biochimica et Biophysica Acta (BBA) - Bioenergetics
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