CO impedes superfast O₂ binding in ba₃ cytochrome oxidase from Thermus thermophilus

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 ba₃ 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 O₂-carrier with a 355 nm laser pulse and the fully reduced CO-bound ba₃ simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O₂ binding to heme a3 at 90 μMO₂ 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 × 10⁹ M^-1 s^-1 for O₂ binding to ba₃ 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 O₂ bond cleavage in ba₃ 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 O₂ to the active site in ba₃ and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a3 site in ba₃, making the CO flow-flash method inapplicable.