Surveying redox regulation of Chlamydomonas reinhardtii Rubisco through directed mutagenesis scanning of conserved cysteine residues
Hemanth Phani K. Sudhani, María Jesús García-Murria, Julia Marín-Navarro, and Joaquín Moreno.
Departament of Biochemistry and Molecular Biology, University of Valencia, Dr. Moliner 50, Burjassot E-46100, Spain
The carbon-fixing enzyme, ribulose 1,5-bisphosphate carboxylase/ oxygenase (Rubisco) from photosynthetic eukaryotes is known to be sequentially inactivated and structurally destabilized by the progressive oxidation of sulfhydryl groups from accesible cysteine residues to disulfides. The critical cysteines involved in this transition have not been identified yet, although the similar sensitivity of all eukaryotic Rubiscos of the green-like type tested so far points to conserved residues. In an attempt to analyze the contribution of the different residues, we have replaced one by one all conserved cysteines by serines (a single-atom substitution that prevents oxidation) through site-directed mutagenesis and chloroplast (for residues 84, 172, 192, 247, 284, 427, 449 and 459 of the large subunit) or nuclear (for residue 83 of the small subunit) transformation of C. reinhardtii. Oxidation-sensitivity analysis of the mutants indicated that the replacement of Cys 172 results in an enzyme that is somewhat more resistant to oxidative destabilization. Cys 449 and 459 are also partially responsible for oxidative inactivation of Rubisco because the absence of these cysteines prevents complete activity loss under strong oxidative conditions. In contrast, substitution of Cys 192 or 427 increases Rubisco sensitivity to oxidation. Cys 84 seems to play a significant role in mantaining the holoenzyme structure both in the reduced and oxidized form. Nevertheless, substitution of single residues could not prevent neither substantial inactivation nor structural destabilization upon oxidation. Therefore, it might be concluded that these drastic changes are brought forth through oxidative modification of a group of critical cysteines with highly redundant roles.
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