Comparative analyses of wild-type XfOhr and two mutants (R19A and E51A).

<p>(A) Lipoamide-lipoamide dehydrogenase peroxidase-coupled assay of wild-type XfOhr, R19A and E51A. The peroxidase activities were monitored by the oxidation of NADH at 340 nm in the presence of XfOhr (0.05 μM), lipoamide dehydrogenase from <i>X</i>. <i>fastidiosa</i> (XfLpD, 0.5 μM), and lipoamide (50 μM) in sodium phosphate buffer (20 mM, pH 7.4) and DTPA (0.1 mM). Cys61 (C_p) pKa determination of wild-type XfOhr and two mutants (R19A and E51A) by the monobromobimane method; plots of fluorescence as a function of pH for wild-type XfOhr (B), R19A XfOhr (C) and E51A XfOhr (D). The red points show the mean values of at least two independent experiments. The error bars indicate the SEM. All pKa values were determined using the Henderson-Hasselbach equation of GraphPad^®Prism4.