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 ...

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.

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates their functions. Using photolabile NO and O2 carriers, we recently found that NO and O2 binding in Thermus thermophilus ba3 (Tt ba3) is ~10-times faster than in the bovine enzyme, indicating inherent structural differences that affect ligand access in these enzymes. Using x-ray crystallography, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in the Tt ba3 mutants Y133W, T231F, and Y133W&T231F, in which tyrosine and/or threonine in the O2-channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine present in the aa3 enzymes. NO binding in Y133W and Y133W&T231F is 5-times slower than in wild-type ba3 and the T231F mutant, and 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 “way-station,” which may further slow ligand access to the active site. Classical simulations of Xe diffusion 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.

The route of O2 to and from the high-spin heme in heme–copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to CuB+ on the pathway to and from the high-spin heme. The presence of CO on CuB+ suggests that O2 binding may be compromised in CO flow-flash experiments. Time-resolved optical absorption studies show that the rate of O2 and NO binding in the bovine enzyme (1 × 108 M−1 s−1) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t1/2 = 1.5 μs) of CO from CuB+. In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O2 and NO binding to heme a3 slows by an order of magnitude in the presence of CO (from 1 × 109 to 1 × 108 M−1 s−1), but is still considerably faster (~10 μs at 1 atm O2) than the CO off-rate from CuB in the absence of O2 (milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O2 and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on CuB+ impedes the binding of O2 to CuB+ or, if O2 does not bind to CuB+ prior to heme a3, whether the CuB+-CO complex sterically restricts access of O2 to the heme. Both possibilities are discussed, and we argue that O2 binds directly to heme a3 in Tt ba3, causing CO to dissociate from CuB+ in a concerted manner through steric and/or electronic effects. This would allow CuB+ to function as an electron donor during the fast (5 μs) breaking of the O–O bond. These results suggest that the binding of CO to CuB+ on the path to and from heme a3 may not be applicable to O2 and NO in all heme-copper oxidases. This article is part of a Special Issue entitled: Vibrational Spectroscopies in Molecular Bioenergetics.

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617–11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.