Figure 5.

Reduction of compound I by sulfide. (A) Spectral changes upon addition of 5 µM sulfide to 2 µM compound I in a sequential-mixing stopped-flow experiment. Compound I was formed by mixing 4 µM MPO with 40 µM H₂O₂ using a delay time of 20 ms. First spectrum was recorded 9 ms after the second mixing cycle and the subsequent spectra as indicated in the figure. Arrows show the direction of the spectral transitions. (B) Typical biphasic kinetic trace (black line) with a double-exponential fit (red line) showing the reaction of compound I with 5 µM sulfide followed at 456 nm. (C) Dependence of the obtained pseudo first-order rate constants, obtained from the first (green dots) and second (purple dots) phases of the double-exponential kinetic traces upon compound I reduction, on the sulfide concentration. (D) Spectral changes after mixing 2 µM compound I with 20 µM sulfide in a sequential-mixing stopped-flow experiment. Reaction conditions: 100 mM phosphate buffer, pH 7.4, and 25°C.