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/H(2)O(2) 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 Fe(2+) 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 A. Furthermore, their C(alpha)H 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 rbcL 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.
Luo
Shen
S
Department of Applied Plant Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan.
Ishida
Hiroyuki
H
Makino
Amane
A
Mae
Tadahiko
T
D001665:Binding Sites; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D007501:Iron / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014908:Triticum / Q000378:metabolism
D006861:Hydrogen Peroxide; D007501:Iron; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium; D010100:Oxygen