Figure 5.

Direct observation of hyperoxidation kinetics with C171A mutants. (a) Pre-steady-state kinetics for the reaction of Tsa1^C171A (5 μM) with H₂O₂ (25, 50, 200, 400, and 800 μM as indicated) monitored by Trp fluorescence, fitted against a biexponential equation (red lines). Phase 4 is labeled. The * indicates a small, faster phase that is not attributed. (b) Second-order plot and linear fit of the slow phase observed rate constant k₄^obs (deduced from panel (a) fits) against H₂O₂ concentration, for Tsa1^C171A (black circles, black fit), Tsa1^W161F C171A (black diamonds, black fit), and Tsa1^{C171A A177S A178D} (black squares, red fit). (c) Deconvoluted mass spectra of the Tsa1^C171A redox species before and after 5 s reaction with 200 μM H₂O₂, followed by acid quenching.