Fig. 5.
O2, NO, and CO are regulatory ligands of DosT. (A) Effect of O2 and NO on DosT autokinase activity. To remove bound O2 and generate the deoxy form (Fe2+), oxy DosT (Fe2+−O2) was deoxygenated, transferred to the glove box, exposed to DTH, and analyzed in the autokinase assay. To study the effect of NO on autokinase activity, oxy DosT (Fe2+−O2) and deoxy DosT (Fe2+) was exposed to proline NONOate and are indicated by the Fe2+−O2 +NO and Fe2+−NO labels, respectively. (B) To study the effect of CO on autokinase activity, oxy DosT (Fe2+−O2) and deoxy DosT (Fe2+) were exposed to CO for 30 s and are indicate by the Fe2+−O2 +CO and Fe2+−CO labels, respectively. Note that partial substitution of oxygen bound to DosT by CO (performed in the presence of air) leads to increased autokinase activity, albeit not as much as the heme-carbonyl and deoxy DosT samples.