Mitochondrial ATP synthase: Fe2+-catalyzed fragmentation of the soluble F-1-ATPase

The treatment of the soluble F-1-ATPase with the Fe2+-ascorbate oxidative system has resulted in the inactivation and fragmentation of the enzyme. Up to 10 polypeptide fragments could be readily observed on the SDS-PAGE. Addition of free Mg2+ or EDTA effectively prevented inactivation and fragmentation. Both alpha and beta subunits of the F-1-ATPase were cleaved, with predominant cleavage sites being identified on alpha. Oxidative fragmentation of the F-1-ATPase showed nucleotide dependence. Removal of nucleotides from the F-1-ATPase as well as their excess in the medium dramatically affected the fragmentation pattern. On the basis of the M(r) of the fragments, their immunorecognition with the antibodies against subunits of the F-1-ATPase, and the results of the mild proteolysis of the F-1-ATPase with trypsin, cleavage sites are suggested to be located in the nucleotide-binding domain of both alpha and beta subunits. Finally, it is hypothesized that similar structural damage of the F-1-ATPase may occur in mitochondrion in vivo under oxidative stress conditions. (C) 1996 Academic Press, Inc.