Figure 4.

Absorbance changes after flash photolysis of CO from the fully reduced enzyme in the presence of O₂. (A) Sequence of events in the wild-type (WT) and mutant enzymes. The length of the arrows has been adjusted to approximately fit the time course of the absorbance changes in B–E. At 590 nm (B) the increase in absorbance with τ ≅ 8 μ s is associated with binding of O₂ to reduced heme a₃ (intermediate A). The subsequent decrease in absorbance with τ ≅ 60 μs is associated with the formation of P_r. This kinetic phase is not observed with the SD(I-299)/SG(I-299) mutant enzymes. At 605 nm (C) the redox state, primarily of heme a, is monitored. In both mutant enzymes, heme a is oxidized more slowly than in the wild-type enzyme, and the oxidation coincides with formation of F (increase in absorbance at 580 nm, D). Changes in the redox level of Cu_A contribute significantly at 830 nm (E), where an increase in absorbance corresponds to Cu_A oxidation. The noise level is larger at short times because the traces were recorded on a logarithmic scale. Experimental conditions: 0.1 M Hepes, pH 7.4, 0.1% dodecyl-β-d-maltoside, ≈2 μM reacting enzyme (all traces have been normalized to 1 μM reacting enzyme), 1 mM O₂, 22°C.