Figure 4.

Steady-state hyperoxidation sensitivity of wild-type and mutant Tsa1 with H₂O₂. (a) Steady-state kinetics for the determination of hyperoxidation sensitivity of Tsa1 monitored by consumption of NADPH (200 μM) at 340 nm in the presence of thioredoxin reductase (0.25 μM), Trx (5 μM), Tsa1 (1 μM), and variable amounts of H₂O₂ (from 50, 100, 150, 200, 300, etc. to 1 mM) in TK buffer. The time courses have been shifted on the y axis for clarity. (b) Secondary plot of the inactivated fraction f_inact per turnover deduced from (a) vs H₂O₂ concentration. The hyperoxidation index C_hyp1% is deduced from the slope of the linear fit for wild type (black circles, black line fit), mutants Tsa1^W161F (black diamonds, blue line fit) and Tsa1^A177S A178D (black squares, red line fit), and hPrx1 (black triangles, purple line fit). Data are the mean of two independent experiments. (c) Pre-steady-state kinetics for the reaction of Tsa1^A177S A178D, Tsa1, or Tsa1^W161F (5 μM, top to bottom) with H₂O₂ (10 μM) monitored by Trp fluorescence as in Figure 2b, fitted against a three-exponential equation (red or black line). Time courses have been shifted on the y axis for clarity. (d) Far-UV CD spectra of 5 μM Tsa1 (black), Tsa1^W161F (red), Tsa1^A177S A178D (blue), and Tsa1^Y190G F191G (green) under the reduced state. Measurements were performed in a 0.01 cm flat cell in phosphate (10 mM) NaF (100 mM) buffer (pH 7) and are the average of three records.