Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site.

We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide−thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the Vmax for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1. Upon chelation of Fe3+ ions to the Cys 540-bound EDTA and incubation of the S-1 derivative−Fe complex with ascorbic acid at pH 7.5, the 95 kDa heavy chain underwent a conformation-dependent, single-cut oxidative fragmentation within 5−15 A of Cys 540. Three pairs of fragments were formed which, after specific fluorescent labeling and SDS−PAGE, could be positi...