Fe2+-catalyzed site-specific cleavage of the large subunit of ribulose 1,5-bisphosphate carboxylase close to the active site

Previous work has demonstrated that the large subunit (rbcL) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from wheat is cleaved at Gly-329 by the Fe (2+)/ascorbate/H2O2 system (Ishida, H., Makino, A., and Mae, T. (1999) J. Biol Chem. 274, 5222-5226). In this study, we found that the rbcL could also be cleaved into several other fragments by increasing the incubation time or the Fe2+ concentration. By combining immunoblotting with N-terminal amino acid sequencing, cleavage sites were identified at Gly-404, Gly-380, Gly-329, Ala-296, Asp-203, and Gly-122. Conformational analysis demonstrated that five of them are located in the alpha/beta-barrel, whereas Gly-122 is in the N-terminal domain but near the bound metal in the adjacent rbcL. All of these residues are at or very close to the active site and are just around the metal-binding site within a radius of 12 Angstrom. Furthermore, their C H-alpha groups are completely or partially exposed to the bound metal. A radical scavenger, activation of RuBisCo, or binding of a reaction-intermediate analogue to the activated RuBisCo, inhibited the fragmentation. These results strongly suggest that the rbeL is cleaved by reactive oxygen species generated at the metal-binding site and that proximity and favorable orientation are probably the most important parameters in determining the cleavage sites.