CO impedes superfast O ₂ binding in ba ₃ cytochrome oxidase from Thermus thermophilus

Abstract

Kinetic studies of heme-copper terminal oxidases using the CO flow-flash method are potentially compromised by the fate of the photodissociated CO. In this time-resolved optical absorption study, we compared the kinetics of dioxygen reduction by ba 3 cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile O 2 -carrier. A novel doublelaser excitation is introduced in which dioxygen is generated by photolyzing the O 2 -carrier with a 355 nm laser pulse and the fully reduced CO-bound ba 3 simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O 2 binding to heme a 3 at 90 μM O 2 occurs with lifetimes of 9.3 and 110 μs in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 μs), which generates the P intermediate with the concomitant oxidation of heme b. The second-order rate constant of 1 × 10 9 M −1 s −1 for O 2 binding to ba 3 in the absence of CO is 10 times greater than observed in the presence of CO as well as for the bovine heart enzyme. The O 2 bond cleavage in ba 3 of 4.8 μs is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of O 2 to the active site in ba 3 and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a 3 site in ba 3 , making the CO flow-flash method inapplicable.double-laser technique | T. thermophilus ba3 | oxygen reduction | slow-fast kinetics | O2 channel T he reduction of dioxygen to water in the heme-copper oxidases takes place at the high-spin heme a 3 and Cu B heterodinuclear center (for review, see refs. 1 and 2). The reaction has been extensively investigated in several aa 3 -oxidases by timeresolved spectroscopic techniques in combination with the CO flow-flash technique (1, 2), in which the reaction is initiated by photolyzing CO bound to heme a 3 2þ in the presence of O 2 (3). The O 2 reduction has commonly been interpreted in terms of a unidirectional sequential mechanism (Scheme 1).The O 2 reduction in Thermus thermophilus ba 3 , a B-type oxidase with distant sequence homology to the A-type oxidases (4), has received much less attention (5-7). The enzyme contains the four redox-active metal centers (8-10) and functions as a terminal oxidase for aerobic metabolism under limited oxygen concentration (8-11). It also possesses NO reductase activity (12) suggesting shared evolutionary lineage of O 2 ∕NO reduction in this enzyme. In ba 3 , the thermal dissociation of CO from heme a 3 2þ in the dark is significantly faster (0.8 s −1 ) (5) than in the bovine aa 3 (0.023 s −1 ) (3, 13), and therefore CO flow-flash experiments on ba 3 require fast mixing; such experiments have recently been reported (6, 7). Moreover, the Cu B þ -CO complex formed following CO photolysis from heme a 3 2þ in ba 3 decays with a lifetime of approximately 30 ms (14), a rate much slower than that of O 2 binding to heme ...

Adaptation of aerobic respiration to low O 2 environments

“…The maximum turnover of the oxygen reductases is typically in the range of 100-1;000 electrons∕s. If the second order rate constant for the initial reaction of O 2 with the enzyme is 10 8 M −1 s −1 [the value measured for the A-family (36)], then at 10 −9 M O 2 the formation of the initial complex between the enzyme and oxygen will be rate limiting at about 10 −1 s −1 . Even though the O 2 concentration within the hydrophobic lipid membrane is higher than in water, the rate of respiration might be too slow to maintain the necessary membrane potential.…”

Probing Protonation/Deprotonation of Tyrosine Residues in Cytochrome ba3 Oxidase from Thermus thermophilus by Time-resolved Step-scan Fourier Transform Infrared Spectroscopy

“…This raises important issues as to the means by which the electron transfer to these transient species is regulated for conformational transitions that are required for any pumping mechanism to occur (6,7,9,10). In the oxidase/peroxide reaction at high pH, it has been demonstrated that the addition of stoichiometric amounts of H 2 O 2 to oxidized enzyme leads to the formation of the 607 nm form having the proposed Fe(IV)ϭO .…”

Spectroscopic and Kinetic Investigation of the Fully Reduced and Mixed Valence States of ba3-Cytochrome c Oxidase from Thermus thermophilus

“…Electron Transfer in Cytochrome c Oxidase-Cytochrome c oxidase utilizes a complex pattern of ET through strongly coupled substrate and redox-dependent sites that involve intermediate cofactors and nonadiabatic, long distance, single-electron tunneling between weakly coupled redox centers (5,11,45,46,66). In the aerobic electron transfer chain of T. thermophilus, electrons are donated by cytochrome c 552 and enter ba 3 -cytochrome c oxidase through the dinuclear Cu A site (39,58,59,67). The interactions between this pair were found to be mainly hydrophobic (68), in contrast to other oxidases where an electrostatic character was described.…”