The tert-butyl hydroperoxide-induced oxidation of actin Cys-374 is coupledwith structural changes in distant regions of the protein

The susceptibility of monomeric actin to both methionine and cysteine oxida tion when treated with the oxidizing agent tert-butyl hydroperoxide (t-BH) was investigated. The results show that no methionine residue was susceptib le to oxidation by t-BH at concentrations of 1-20 mM, while Cys-374, one of the five cysteine residues of the actin molecule, was found to be the site of the oxidative modification. Perturbations in the intrinsic tryptophan f luorescence and the decreased susceptibility to limited proteolysis by cl-c hymotrypsin and subtilisin of oxidized actin give an indication of some alt erations in protein conformation in subdomain 1, and in the central segment of surface loop 39-51, in subdomain 2. Urea denaturation curves indicate a lower conformational stability for the oxidized actin, G-actin structural alterations due to Cys-374 oxidation produced by t-BH result in a decrease in the maximum rate of polymerization, an increase in both the delay time a nd the time required for half-maximum assembly, a decrease in the elongatio n rate, and enhancement of the critical monomer concentration for polymeriz ation. The results suggest that oxidation of actin Cys-374 induces structur al alterations in the conformation of at least two different distant region s of the molecule. The involvement of both the C-terminus of the actin poly peptide chain and the DNase-I-binding loop in the intermonomer interactions in the polymer could account for the altered kinetics of polymerization sh own by the oxidized actin.