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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 O2-carrier. A novel double-laser 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 μM O2 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.