Figure 2.

Time-resolved absorption changes upon reduction by 1-MNA^∗ radicals. Concentrations: WT P. aeruginosa-NiR 9.6 μM, 5 mM 1-methyl nicotinamide, 5 mM phosphate, 0.1 M tert-BuOH, argon saturated, pH 7.0. Optical path length 3.0 cm, pulse width 0.1 μs, temperature 11.2°C. Measurements were performed at 551 nm, monitoring heme-c reduction (left panel: fast time scale) and heme-c reoxidation (right panel: slow time scale), and at 640 nm, monitoring heme-d₁ reduction (left panel: fast time scale) and heme-d₁ reoxidation (right panel: slow time scale). (A) WT initial pulses after <0.5 reduction equivalents were added. (B) WT after addition of ∼1.1 reduction equivalents were added. (C) WT after addition of ∼2.1 reduction equivalents were added. Concentrations: H369A P. aeruginosa-NiR mutant 48 μM, 2 mM 1-methyl nicotinamide, 10 mM phosphate, 0.1 M tert-BuOH, argon saturated. pH 7.0 Optical path length 3.0 cm, pulse width 1.0 μs; temperature 25.0°C. Measurements were performed at 551 nm, monitoring heme-c reduction (left panel: fast time scale) and heme-c reoxidation (right panel: slow time scale) and at 460 nm, monitoring heme-d₁ reduction (left panel: fast time scale) and heme-d₁ reoxidation (right panel: slow time scale). (D) H369A. Initial pulses after <0.5 reduction equivalents were added. (E) H369A, after addition of ∼1.1 reduction equivalents were added. (F) H369A, after addition of ∼2.1 reduction equivalents were added. Note that, in the case of the mutant, the rate of the faster phase at 551 nm is greater than that with the WT, consistent with the higher enzyme concentration employed.