Cysteines 449 and 459 modulate the reduction-oxidation conformational changes of ribulose 1.5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress.
The role of cysteines 449 (Cys449) and 459 (Cys459) from the large subunit (LS) of ribulose 1-5-bisphosphate carboxylase/oxygenase (Rubisco) in the reduction-oxidation (redox) regulation of the enzyme was assessed by site-directed mutagenesis of these residues and chloroplast transformation of Chlamydomonas reinhardtii. In vitro studies indicated that mutations C449S, C459S or C449S/ C459S do not affect the activity and proteolytic susceptibility of the enzyme in the reduced state. However, when oxidized, the mutant enzymes differed from the wild type (WT), showing an increased resistance to inactivation and, in the case of the double mutant (DM), an altered structural conformation as reflected by the kinetics of proteolysis with subtilisin. The response of the DM strain to saline stress revealed that the absence of Cys449 and Cys459 intensifies Rubisco degradation and the covalent disulfide and non-disulfide polymerization of the enzyme in vivo. Saline stress also induced Rubisco translocation to a membrane (M) fraction that contained only covalently polymerized enzyme. Rubisco mobilization to this M fraction was enhanced also in the DM strain. Altogether, these results indicate that Cys449 and Cys459 participate in the modulation of the conformational changes promoted by oxidative modifications retarding processes related to the catabolism of the enzyme in vivo.
Marín-Navarro
Julia
J
Departament de Bioquimica i Biologia Molecular, Universitat de València, Dr Moliner 50, Burjassot E46100, Spain.
Moreno
Joaquín
J
D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D021381:Protein Transport; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000378:metabolism
D012273:Ribulose-Bisphosphate Carboxylase; D003545:Cysteine