Oxidizing substrate of PrxQ.

<p>(<b>a</b>) The Michaelis-Menten plots of PrxQ activity versus different substrates by NADPH-coupled spectrophotometric method.The reaction mixtures containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM EDTA, 250 μM NADPH, 1 μM PrxQ, 15 μM TrxR, and 40 μM Trx1 and 0–2 mM peroxides (upper left), or 40 μM Trx2 and 0–2 mM peroxides (upper right). The data were analyzed by nonlinear regression using the program GraphPad Prism 5 and were presented as means of the values obtained from three independent assays. (<b>b</b>) Kinetics of peroxynitrite reduction by PrxQ. Peroxynitrite (1 μM) in 10 mM NaOH was rapidly mixed with HRP (5 μM) in the absence or presence of increasing concentrations of PrxQ in 100 mM sodium phosphate buffer (pH 7.4) at 25°C. The inset shows the experimental traces corresponding to HRP compound I formation without PrxQ (control) and with different concentration of PrxQ (0.0, 5.0, 7.5 μM) (lower left). Experimental data were fitted to single exponentials from which observed rate constants of HRP compound I formation were determined. The latter were plotted against PrxQ concentrations (lower right). The data were presented as means of the values obtained from three independent assays.