An intra‐dimeric crosslink of large subunits of spinach ribulose‐1,5‐bisphosphate carboxylase/oxygenase is formed by oxidation of cysteine 247

Abstract

Crystals of the hexadecameric form of ribulose-bisphosphate carboxylase used to solve the structure of the enzyme are composed of protein substantially crosslinked by a disulfide bond between pairs of large subunits. Conditions leading to the selective formation of dimers of the large subunits are described. The stability and specificity of the intra-dimeric crosslink was used to confirm that only one cysteine residue, Cys247 of neighboring large subunits, is involved in the bridge. The ability to generate this disulfide selectively, or alternatively replace the cysteine by site-directed mutagenesis, has led us to conclude that there is no effect of these changes on any of the critical kinetic parameters of the enzyme. The benign effect of the oxidation indicates that the crystal structures of the ribulose-bisphosphate carboxylase, particularly of the active site, are a true representation of the native enzyme.Recent crystallographic studies of the hexadecameric L,S,-type ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu(l ,5)P,ase] from cyanobacteria (Newman and Gutteridge, 1990) have indicated that the large subunits (L) in the crystal structure are covalently linked by a disulfide bridge so as to form large-subunit dimers ( L J . Higher-order structures were not seen. Disulfide-linked large-subunit dimers were also detected in crystals of spinach Rbu(1 ,5)Pzase.

Dual Role of Cysteine 172 in Redox Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activity and Degradation

“…The proximity of C172 to C192 (29) suggests that the two molecules may form an intramolecular disulfide bond. Furthermore, a disulfide bridge between Cys247 residues in the large subunit homodimer has been shown to be present after enzyme oxidation (30). Therefore, we examined the effect of replacement of Cys and whether the redox state affects the thermal stability of the enzyme by analyzing carboxylase activity after incubation at temperatures ranging from 5 to 100°C.…”

“…The RuBisCO from cyanobacteria, algae (excluding some dinoflagellates), and higher plants is a hexadecamer consisting of eight large and eight small subunits organized as four protomers, each of which has two active sites at the interface between each large subunit pair (3,4,29). In the RuBisCO of many species, an intradimeric disulfide bond is formed under oxidizing conditions between the Cys247 residues of a large subunit pair (30). A sequence comparison of more than 500 RuBisCO genes revealed high levels of similarity, particularly among large subunits, and residues that form the catalytic site are entirely conserved (21).…”

“…However, no direct evidence for the existence of a disulfide bond between these residues has been obtained thus far. Besides this pair of cysteine residues, each pair of large subunits may cross-link via a Cys247-Cys247 disulfide bond (30).…”

C172S Substitution in the Chloroplast-encoded Large Subunit Affects Stability and Stress-induced Turnover of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

“…This indicates that the increase in thermal sensitivity is due to DTT acting on disulfides other than the Cys-172/Cys-192 pair. Nonetheless, one may argue that the putative Cys-172-Cys-192 disulfide bond is resistant to DTT, as is the Cys-247-Cys-247 disulfide bond that is buried within the hydrophobic core of spinach Rubisco (7,44).…”

Crystal structure of activated tobacco rubisco complexed with the reaction‐intermediate analogue 2‐carboxy‐arabinitol 1, 5‐bisphosphate

“…Although rubisco is not regulated this way (Campbell & Ogren, 1990), it is conceivable that the same regulatory mechanism causes disulfide links to be formed in the dark that are of no consequence for the catalytic activity. Ranty et al (1991) showed that the disulfide link present between dimer-related Cys 247 residues does not influence the catalytic activity. This disulfide link is present in the Data collection crystal structure ofspinach rubisco , Synechococcus rubisco (Newman & Gutteridge, 1990), and tobacco rubisco.…”