FIG. 4.

AMS labeling of Cys thiols of purified apoFNR and FNR from aerobically or anaerobically grown E. coli CAG627(pMW32). Purified apoFNR was reduced with excess DTT (+ DTT) and subsequently labeled with AMS (8 mM) (+ AMS) (lane 1). Lane 2 contains purified unlabeled apoFNR for comparison. Reconstituted [4Fe-4S]FNR was labeled with AMS either directly (lane 3) or after incubation under air for 20 min (lane 4). For labeling in vivo, E. coli CAG627(pMW32) was grown under inducing conditions under anoxic (N₂) (lane 5) or oxic (O₂) (lane 6) conditions to an OD₅₇₈ of 0.8, permeabilized with chloroform, and incubated with 10 mM AMS for 5 min. After the reaction was stopped with the SDS sample buffer, the isolated proteins (2 to 5 μg of purified FNR) and the cellular proteins (30 μg) were separated by SDS-PAGE, blotted onto nitrocellulose, and used for immunoblotting. The positions of proteins corresponding to M_r 27,000, 29,000, and 34,000 are shown.