FIG. 2.

Kinase activities of the DTT-reduced ferrous DevS derivatives. Purified DevS was treated with 10 mM DTT and sparged with N₂ gas for 10 min to generate the deoxyferrous form of DevS. Oxy- and NO-ferrous DevS derivatives were prepared as described in Materials and Methods. The autophosphorylation reactions were performed by using 80 pmol each of DevS, C-DevS, and H150A DevS and 200 mM (1,000 ci/mol) ATP in the anaerobic box at RT. At the time points indicated, samples (10 μl) were removed and added to 3 μl of loading buffer to stop the reaction. (A) The phosphorylation of the protein was assayed by SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. (B) Quantitation of the band intensity of phosphorylated DevS was performed with a densitometer program, ImageJ (version 1.37), and the relative values are plotted as a function of the reaction time. Symbols: filled circle, oxyferrous DevS; open circle, deoxyferrous DevS; filled inverted triangle, NO-ferrous DevS.