Increased Sensitivity of Oxidized Large Isoform of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase to ADP Inhibition Is Due to an Interaction between Its Carboxyl Extension and Nucleotide-binding Pocket

Abstract

In Arabidopsis, oxidation of the large (46-kDa) isoform activase to form a disulfide bond in the C-terminal extension (C-extension) significantly increases its ADP sensitivity for both ATP hydrolysis and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation, thereby decreasing both activities at physiological ratios of ADP/ATP. In this study, we demonstrate that the C-extension of the oxidized large activase isoform can be cross-linked with regions containing residues that contribute to the nucleotide-binding pocket, with a higher efficiency in the presence of ADP or the absence of nucleotides than with ATP. Coupled with measurements demonstrating a redox-dependent protease sensitivity of the C-extension and a lower ATP or adenosine 5 -O-(thiotriphosphate) (ATP␥S) affinity of the oxidized large isoform than either the reduced form or the smaller isoform, the results suggest that the C-extension plays an inhibitory role in ATP hydrolysis, regulated by redox changes. In contrast, the ADP affinities of the small isoform and the reduced or oxidized large isoform were similar, which indicates that the C-extension selectively interferes with the proper binding of ATP, possibly by interfering with the coordination of the ␥-phosphate. Furthermore, replacement of conserved, negatively charged residues (Asp 390 , Glu 394 , and Asp 401 ) in the C-extension with alanine significantly reduced the sensitivities of the mutants to ADP inhibition, which suggests the involvement of electrostatic interactions between them and positively charged residues in or near the nucleotide-binding pocket. These studies provide new insights into the mechanism of redox regulation of activase by the C-extension in the large isoform.Rubisco 2 activase, a nuclear-encoded chloroplast protein, facilitates the conversion of Rubisco from an inactive to active form by releasing tightly bound, inhibitory sugar phosphates from the active site (1). This process requires ATP hydrolysis by activase and is inhibited by ADP (2). Activase belongs to an AAA ϩ (ATPase associated with diverse cellular activities) protein family, based on sequence homology of its central portion with common AAA motifs, and each monomer contains one nucleotide-binding pocket consisting of residues from Walker A, Walker B, and Sensor 1 domains (1, 3). Site-directed mutagenesis and photoaffinity labeling (4-6) showed that the Walker A motif (GXXXGKS; P-loop) is involved in nucleotide binding. A conserved aspartate residue in the Walker B motif (hhhhDEXX, h ϭ hydrophobic residue) is involved in metal ligand binding and ATP catalysis (7). The Sensor 1 region has also been implicated in the binding/coordination of ATP (6, 7). Recently, two residues in the Sensor 2 domain were identified as determining specificity for Rubisco (8), in agreement with the involvement of this motif in substrate recognition in other AAA ϩ members. Several residues in Box VII, which is part of the linkage between the Sensor 1 and Sensor 2 motifs, are essential for maintaining a functional enzym...

The Regulatory Properties of Rubisco Activase Differ among Species and Affect Photosynthetic Induction during Light Transitions

“…In wild-type Arabidopsis, b-Rca is paired with a-Rca, and the interaction of the two isoforms affects the sensitivity of the holoenzyme to inhibition by ADP (Zhang et al, 2001;Wang and Portis, 2006). That the Cys residues in the C-terminal extension are conserved in a-Rca from diverse species, including those produced from separate genes (Supplemental Fig.…”

“…Our current understanding of the role of the two Rca isoforms is based primarily on investigations with Arabidopsis (Arabidopsis thaliana; Zhang and Portis, 1999;Zhang et al, 2001;Wang and Portis, 2006). The C-terminal extension of the a-Rca contains two redox-regulated Cys residues that are modulated by thioredoxin f (Zhang and Portis, 1999).…”

Cool C4 Photosynthesis: Pyruvate Pi Dikinase Expression and Activity Corresponds to the Exceptional Cold Tolerance of Carbon Assimilation inMiscanthus×giganteus

“…The blots were incubated with fluorescent-conjugated rabbit secondary antibodies (IRdye; LI-COR). The polyvinylidene difluoride membranes were scanned by using an Odyssey Infrared Imaging system (LI-COR), and relative protein expression levels were quantified with the Odyssey V1.2 application software as described previously (Wang and Portis, 2006).…”

NanoESI Mass Spectrometry of Rubisco and Rubisco Activase Structures and Their Interactions with Nucleotides and Sugar Phosphates

“…These observations support the assignment of oligomeric forms in Figure 1b; however, the ions from hexamer+nADP were too low in abundance to assign with certainty. While prior studies using fluorescence quenching with 1-anilinonaphthalene-8-sulfonic acid and intrinsic fluorescence have been used to examine differences in the nucleotide binding capacity of modified and unmodified RA [42,43], our nanoESI-MS measurements uniquely demonstrate the capacity of each RA monomer to bind ADP.…”

“…Based on the abundance of ions from free and ADP-bound RA, ≥50% of the purified RA was present in a complex with ADP. Importantly this may influence the capacity of RA to oligomerize given the inhibitory influence of bound ADP on the ATPase and Rubisco activation activities of RA and the stimulatory effect of ATP on RA self-association [13,15,22,24,42,44]. Therefore, in order to be able to examine the effects of added nucleotides on the oligomerization of RA and to compare binding affinities of nucleotides by nanoESI-MS, a method was developed to remove bound ADP from RA.…”