Fig. 2.
Reaction of the fully reduced wild type and E25P variant cbb3s with O2. A laser flash initiates the reaction at t = 0. Shown are the absorbance changes at (A) 420, (B) 430, (C) 550 (reporting oxidation of the c-type hemes) and (D) 560 nm (reporting on the low-spin heme b). Experimental conditions (after mixing): approximately 1 μM cbb3, 50 mM Hepes pH 7.5, 50 mM KCl, 0.03% DDM, [O2] = 1 mM, T = 298 K. The traces have been normalized using the initial absorbance change (from the CO dissociation) at 420 nm. E shows proton uptake from solution during the reaction of fully reduced wild type and E25P variants with O2. Experimental conditions: approximately 1 μM cbb3, 50 mM KCl, 50 μM EDTA, 0.03% DDM, 40 μM phenol red, [O2] = 1 mM, T = 298 K. Shown are the differences between the traces obtained in the absence of buffer and the ones obtained after adding 20 mM Hepes, pH 7.5. The traces for the different cbb3 variants have been normalised to the same absorbance change after 800 ms to emphasize the difference in rates.