Characterization of the ferrous ion binding sites of apple 1-aminocyclopropane-1-carboxylate oxidase by site-directed mutagenesis.

The putative ferrous ion binding sites (H177, D179, and H234) of apple fruit 1-aminocyclopropane-1-carboxylate oxidase determined by homology comparisons with enzymes which required non-haem Fe2+ for activity were altered by site-directed mutagenesis. The activities of mutants (H177F, D179H, D179A, H234F, and H234D) were completely lost. SDS-PAGE and western immunoanalysis confirmed that loss of enzyme activity in mutants was not due to impaired enzyme expression. These results strongly suggest that H177, D179, and H234 are the Fe2+ binding site.