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

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 1×109 M−1 s−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 italica32+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.

Design of turbulent tangential micro-mixers that mix liquids on the nanosecond time scale

“…The great majority of proteins are stable at these pressures [47], further indicated by the general experience that these HPLC pumps can be used for purification of native active proteins and enzymes by column chromatography. In our freeze-quench experiments using the same HPLC setup and stainless-steel mixer described here, we have never encountered denaturation problems and the rate constants determined at 20 to 40 MPa by us [28,43,44,48,49] are similar to those determined by others working at ambient pressures [5,6,8,12]. In general, oligomeric proteins start to dissociate at pressures of 100 to 200 MPa, and monomeric proteins denature at approximately four times higher pressures [47].…”

“…The time resolutions for turbulent mixing and subsequent observation in stopped-flow ($0.5-1 ms [5,6]) and continuousflow ($11-50 ls [7][8][9]) mixing instruments are much lower than those for relaxation techniques. The shortest time scale for protein and RNA folding or breaking and making chemical bonds in reactions catalyzed by enzymes is 0.1 to 1 ls [1,2,[8][9][10][11][12][13][14] and, thus, is not accessible with mixing techniques. Fast enzymes with kinetic phases on the (sub)microsecond time scale include many members of the oxidoreductases (e.g., catalase, cytochrome oxidases), hydrolases (e.g., acetylcholinesterase), and lyases (e.g., carbonic anhydrase) but are also found within the isomerases and ligases.…”

The cytochrome ba3 oxidase from Thermus thermophilus does not generate a tryptophan radical during turnover: Implications for the mechanism of proton pumping

“…The electrons are transferred from Cu A at the docking site via the low-spin heme b to the high-spin heme a 3 -Cu B binuclear center. When the fully (four-electron) reduced enzyme is exposed to oxygen at neutral pH, the electron transfer reactions following binding of oxygen depicted in Scheme 1 are observed [2][3][4]:…”

“…The rates of the reactions are estimated at k a = 100,000 s −1 , k b = 200,000 s −1 , k c = 18,000 s −1 and k d = 1000 s −1 , with [O 2 ] = 90 μM for the reaction at ambient temperature (20°C-25°C) [2,3] . These rates were observed in time-resolved spectroscopic studies using photolabile O 2 carriers in the absence of CO.…”

“…These rates were observed in time-resolved spectroscopic studies using photolabile O 2 carriers in the absence of CO. In contrast, the rate of CO dissociation has been shown to limit the rate of oxygen binding dramatically in flow-flash studies on cytochrome ba 3 oxidase [2,3], as a result of which the kinetics of oxygen binding and O-O bond breaking are usually not resolved [4][5][6][7] although they apparently were in [8].…”