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ABB Archives of Biochemistry and Biophysics 414 (2003) 150–158 www.elsevier.com/locate/yabbi Minireview The life of ribulose 1,5-bisphosphate carboxylase/oxygenase—posttranslational facts and mysteriesq Robert L. Houtza and Archie R. Portis Jr.b,* a b Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, N322D Agricultural Science Center North, University of Kentucky, Lexington, KY 40546-0091, USA Photosynthesis Research Unit, Agricultural Research Service, United States Department of Agriculture and Department of Crop Sciences and Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA Received 13 December 2002, and in revised form 26 February 2003 Abstract The life of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), from gene to protein to irreplaceable component of photosynthetic CO2 assimilation, has successfully served as a model for a number of essential cellular processes centered on protein chemistry and amino acid modifications. Once translated, the two subunits of Rubisco undergo a myriad of co- and posttranslational modifications accompanied by constant interactions with structurally modifying enzymes. Even after final assembly, the essential role played by Rubisco in photosynthetic CO2 assimilation is dependent on continuous conformation modifications by Rubisco activase. Rubisco is also continuously assaulted by various environmental factors, resulting in its turnover and degradation by processes that appear to be enhanced during plant senescence. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Ribulose 1,5-bisphosphate carboxylase/oxygenase; Posttranslational; Methylation; Protein folding; Ribulose 1,5-bisphosphate; Activase; Oxidation Recognized for decades as a unique catalyst and limiting factor in photosynthetic CO2 assimilation, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco)1 has been the subject of numerous intense scientific studies. This interest has resulted in many pioneering investigations that now serve as models for a variety of cellular mechanisms that are not restricted to plant photosynthetic processes. In this review, we focus on co- and posttranslational modifications that accompany the biochemical life of this enzyme. The diversity of these modifications as they relate to the synthesis, assembly, regulation, and degradation of q This work was supported by U.S. Department of Energy Grants DEFG02-92ER20075 (R.L.H.) and DEAIG2-97ER20268 (A.R.P.). * Corresponding author. Fax: 1-217-244-4419. E-mail address: [email protected] (A.R. Portis Jr.). 1 Abbreviations used: DEF, peptide deformylases; HMT, histone methyltransferase; LS, large subunit; LSMT, LS e N -methyltransferase; PMSR, peptide Met sulfoxide reductase; SS, small subunit; SSMT, SS a N -methyltransferase; Rubisco, ribulose 1,5-bisphosphate carboxylase/ oxygense; RuBP, ribulose-1,5-bisphosphate. Rubisco are summarized in Fig. 1. Many of these structural and conformational changes have been shown to be instrumental in the regulation and assembly of Rubisco but, more importantly, several remain as largely unexplored areas with potential to increase the body of knowledge about this crucial carbon-fixing enzyme. Amino acid modifications Both the large subunit (LS) and the small subunit (SS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) are extensively modified either during and/or after translation. Evidence for these amino acid modifications comes primarily from structural analyses of the mature forms of the polypeptides and subsequent comparison with the known DNA sequences. In some cases, this evidence is corroborated by the characterization of specific enzymes responsible for these modifications. 0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00122-X R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158 Fig. 1. Schematic summary of various co- and posttranslational modifications of Rubisco that are currently known to occur during the three phases of its life cycle. Some modifications are species specific. SS modifications Prior to import into the chloroplast, the N-terminal transit sequence of the SS is phosphorylated (probably at Ser-21) as part of a process that partially regulates import [1]. During or immediately after import, the transit sequence is removed by a stromal processing peptidase in what appears to be a two-step proteolytic process [2–6]. The final processing exposes a conserved Met residue, which is methylated to form an a N -methylmethionine [7,8]. This is a widespread modification found in the SS of Rubiscos from a number of species [7,9–11]. The enzyme responsible for this modification, Rubisco SS a N -methyltransferase (SSMT) [11], is related to the LS e N -methyltransferase (LSMT) responsible for the methylation of Lys-14 in the LS. Although the functional significance of this modification is unknown, it would provide a mechanism for differentiating between N-terminal Met residues on imported proteins and proteins translated in the chloroplast and may provide protection against aminopeptidases. LS modifications The N-terminal residue of the LS of Rubisco in all higher-plant species examined to date is acetylated Pro-3 [12–14]. This region of Rubisco is not discernible by Xray crystallography, and identification has been exclusively by mass spectroscopy of proteolytically removed N-terminal fragments. Comparison of the N termini of the LS with the predicted translation product from the DNA sequence clearly indicates a number of co- and posttranslational modifications. First, protein translation in the chloroplast is initiated with N -formylmethionine [15], and the LS of Rubisco likely begins with this residue. During translation deformylation of N -formyl- 151 methionine occurs by chloroplast-specific peptide deformylases (DEF) (EC 3.5.1.88) [16–18]. As in prokaryotic organisms, this is an essential process [16,17,19] and one upon which all subsequent N-terminal processing is dependent. A biochemical characterization and determination of polypeptide substrate specificity of chloroplast DEFs has been reported. The data suggest that the two chloroplast-localized DEFs have different polypeptide substrate specificities and kinetic parameters [17,19,20]. Shortly after removal of the N -formyl group, Met-1 is removed, probably as a result of N-terminal processing by chloroplast-specific Met aminopeptidases [16,18]. Transcripts that code for such peptidases with predicted chloroplast-targeting sequences have been reported in Arabidopsis, but direct detection and characterization of these enzymes remains to be shown [18]. Although acetylated Ser-2 was reported earlier as the N-terminal residue of the LS [21], the structural identification of the N terminus of the LS of Rubisco from a wide range of species suggests that further N-terminal processing results in the removal of Ser-2 and subsequent acetylation of Pro-3 [13]. Whereas amino peptidases have been reported in the chloroplast, none have been described with specificity for N-terminal Ser residues [22]. How Ser-2 is removed remains to be described. Alternatively, removal of Met–Ser as a dipeptide could occur. The remaining N-terminal Pro residue is acetylated and this could provide protection against proteolytic attack since Prospecific iminopeptidases have been reported in both leaves and chloroplasts [22,23]. Na -acetylation is one of the most common cotranslational protein modifications, perhaps occurring on 80% of all proteins [24–26]. Genes encoding N a -acetyltransferases have been sequenced and characterized from Saccharomyces cerevisiae [27– 30] and Trypanosoma brucei [31]. At least in two reports, the activity of these enzymes has been shown to be essential. To date, however, no chloroplast-localized N a acetyltransferases have been described or annotated in plant genome sequences. In some but not all plant species, the LS is additionally modified by trimethylation of the e-amino group of Lys-14 to form a trimethyllysine residue [12,13]. The modification is common in the LS of Rubisco from Solanaceae and Cucurbitaceae species, but there is still not enough information to establish any definitive species correlation with the absence or presence of a trimethyllysine residue. The temporal nature of this modification is not known, but studies have clearly demonstrated that this modification can occur posttranslationally even after holoenzyme assembly [32,33]. The enzyme responsible for this modification is a Rubisco LSMT (EC 2.1.1.127) [34–36]. Although many different proteins contain trimethyllysine residues, each is associated with a specific protein e N -methyltransferase [37]. The protein e N -methyltransferase responsible for 152 R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158 the methylation of Lys-14 in the LS of Rubisco has been thoroughly characterized. A high-resolution crystal structure for pea Rubisco LSMT which identified a novel bipolar arrangement of substrate binding sites was recently obtained. One binding site has specificity for the N-terminal region of the LS of Rubisco and the other for S-adenosylmethionine, with a pore through which methyl group transfer occurs connecting the two sites [38]. Models describing the docking of Rubisco LSMT to Rubisco suggest that extensive regions of Rubisco LSMT are involved in polypeptide recognition and that the molar stoichiometry of Rubisco LSMT binding to Rubisco is 4 to 1 (Fig. 2). The substrate binding sites are located in a conserved polypeptide sequence (SET domain) with secondary structural homology to a large number of highly evolutionarily conserved proteins known to influence gene expression [39,40]. Many of these proteins have been recently identified as histone methyltransferases (HMTs) [41–44]. Moreover, the mechanism for HMT influence on gene expression led to the identification of proteins that are recruited to histone methylation sites through a specific binding domain for trimethyllysine residues known as the chromodomain [45–47]. Since the functional significance of trimethyllysine-14 in the LS of Rubisco remains an enigma, perhaps these recent advances in discerning the functional significance of Lys methylation in histones will provide clues for identification of the functional significance of trimethyllysine residues in Rubisco and may identify chloroplast proteins recruited to the N-terminal region of Rubisco. Recently the structural determination of Chlamydomonas Rubisco revealed the presence of several other amino acid modifications in the LS [9,10]. Both Pro residues 151 and 104 were found as 4-hydroxyproline residues and Cys residues 256 and 369 as S (gamma)methylcysteine residues. These modifications have not been observed in Rubiscos from other species and may be unique to algal forms of Rubisco. Probably the most dynamic amino acid modification in the LS is the carbamylation of the e-amino group of a highly conserved active-site Lys residue [48,49]. The carbamylation of Lys-201 (Lys-191 in Rhodospirillum rubrum) results in the formation of a metal binding site and, after metal addition, a catalytically competent enzyme. Additionally, the carbamate formed on Lys-201 has been proposed as the essential base responsible for proton abstraction from the C3 position of ribulose 1,5bisphosphate (RuBP) and subsequent ene-diol formation [50]. While carbamylation of the isolated enzyme is a facile and spontaneous process in vitro, the carbamylation level of the enzyme in vivo is determined by the concentrations of various effector ligands that bind to the active site and the interaction of Rubisco with Rubisco activase (discussed below). Clearly the LS and SS of Rubisco are subject to a rather large number of both co- and posttranslational modifications. Given the abundance of Rubisco in the biological world, these amino acid modifications may also represent the most abundant forms of these modified amino acids. Folding and assembly Fig. 2. Model indicating the probable binding between pea Rubisco LSMT and spinach Rubisco. The lower half of the spinach Rubisco holoenzyme structure has been removed, leaving the top octamer of four LSs (light green) and four SSs (dark green) to aid in visualization of Rubisco LSMT. Rubisco LSMT is positioned favorably for Lys-14 (gold) methylation, and the AdoMet binding site is occupied by Sadenosyl homocysteine (red). Reprinted from [38] with permission from Elsevier Science. In prokaryotic organisms, newly translated proteins interact in an ATP-dependent reaction with a welldescribed set of chaperones known as DnaK, DnaJ, and GrpE prior to final assembly by GroEL/GroES chaperonins [51]. The model for this interaction involves the association of DnaJ proteins with newly translated polypeptides, recruitment of DnaK proteins to the same polypeptide, and final formation of a tertiary complex with GrpE. These complexes prevent undesirable polypeptide interactions and potential aggregation. Chloroplast homologs of DnaK [52–54], DnaJ [55], and GrpE [55,56] have been described. Whereas the LS of Rubisco has not been found in direct association with these chaperones, other polypeptides that must be imported into the chloroplast have [57,58] including the transit peptide of the SS [59]. Moreover, the SS transit peptide has been shown to interact directly with chloroplast Hsp70 during import and to contain a recognition domain for Hsp70 [59–61]. The complexes formed between the newly translated polypeptides and the DnaK/DnaJ/ R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158 GrpE complexes represent the first step in cooperative protein folding that ends with the release from the DnaK/DnaJ/GrpE complexes to the GroEL/GroES chaperonins. A similar set of reactions probably operates in chloroplasts but with regard to the LS of Rubisco, more information exists at the level of GroEL/GroES folding than at the earlier steps in protein folding. Evidence for chaperonin-mediated protein folding in the chloroplast was found over 2 decades ago when studies revealed that newly synthesized LSs were transiently associated with a large multimeric complex [62] prior to appearance in the holoenzyme through an ATPdependent reaction [63]. It is now well documented that the assembly of Rubisco requires chloroplast homologs of the GroEL and GroES chaperonins [52,64–67] and that many if not all chloroplast-imported proteins interact with these proteins [68]. However, the absence of an in vitro or bacterial expression system for the assembly of higher-plant Rubisco is evidence of the unique nature of the chloroplast chaperonins and the limitations of our current understanding. The GroEL homologs in the chloroplast are represented by two distinct a and b chaperonin 60s with only 50% sequence homology between them [69]. The exact stoichiometry and arrangement of the a and b subunits in the functional tetradecamer is not known, but tetradecamers composed of only b subunits are significantly less effective in refolding dimeric R. rubrum Rubisco in vitro [70]. The chloroplast GroES homologs are as unique as the a and b chaperonins. Chloroplast chaperonin 10 is much larger than other GroES homologs and is comparable to a fusion between two GroES peptides [71,72]. Protein folding by chloroplast chaperonin 60 tetradecamers requires chaperonin 10, but this requirement can be met by mitochondrial or mouse homologs [70]. The unique nature of the chloroplast chaperonins suggests that there may be protein substrate specificity for chloroplast proteins [70,73], and exploitation of recombinantly expressed forms of the chloroplast chaperonins could lead to the identification of conditions capable of catalyzing the folding and assembly of Rubisco in bacterial or in vitro systems. Such a development would represent a significant advancement in efforts to exploit molecular genetic engineering alterations aimed at increasing the efficiency of Rubisco. However, there may be other requirements for the successful folding and assembly of hexadecameric Rubisco, in particular the activity of other enzymes known to influence proper protein folding, such as peptide disulfide isomerase [74,75] and peptidyl-prolyl cis-trans isomerase [76–78]. Ligand binding and allostery Once properly assembled, the activity of LS8 SS8 Rubiscos can be modified by interaction with the sub- 153 strate RuBP and a wide variety of other effector ligands; phosphoglyceric acid and 2-carboxyarabinitol 1-phosphate are particularly important. The interactions are very complex because (a) both the carbamylated and the uncarbamylated forms of the enzyme are involved, (b) binding can induce a conformational change in the enzyme that closes off the active site, offering a potential for hysteresis, and (c) the two active sites in each dimer of large subunits are composed of residues at the Cterminal domain of one subunit and the N-terminal domain of the other subunit. Thus, there is the opportunity for allostery within each dimer and even perhaps between dimers. To a first approximation, the effects of various effectors on Rubisco are largely accounted for by a simple kinetic model in which they bind to both carbamylated and uncarbamylated Rubisco sites in a competitive manner with RuBP at the active site [79]. This model can account for most ‘‘positive’’ and ‘‘negative’’ effectors and order of addition and dilution effects with in vitro assays reported in the early literature. Under steady state conditions with this model, all effectors will increase the Km for RuBP [80]. In vivo, the situation is complicated by the high concentration of Rubisco sites relative to the concentration of RuBP and various effectors, but equations for the activity of Rubisco under these conditions have been derived [81]. However, the binding of RuBP and various effector ligands is further complicated by a tight binding mode at the active site. The large number of Rubisco crystal structures provides a molecular rationale for the tight binding mode because they form two groups corresponding to either an open or a closed active site [82]. The active site is located at the face of an a=b barrel formed by the C-terminal domain of the LS. The loops connecting the b strands to the a helices contribute the majority of the residues at the active site, but some are also contributed by the N-terminal domain on the neighboring LS. In the closed structures, the ligand at the active site is sequestered from the solvent by loop-6 and the C terminus. Compared to the open structures, the P1 to P2 phosphate distance is reduced in the closed structures, and the N-terminal domain in the neighboring LS is positioned closer, with little change in the positions of the a=b barrels. When the active site is carbamylated and occupied by RuBP, C–C cleavage during catalysis appears to be sufficient to reopen the site, but otherwise release of the tightly bound ligand associated with the ‘‘closed’’ structures requires movement of these domains. A reduction in the catalytic activity of Rubisco due to tight RuBP binding to the uncarbamylated enzyme [83,84] and 2-carboxyarabinitol 1-phosphate binding to the carbamylated enzyme have been observed in vivo [85,86]. In vitro, activity of the enzyme also slowly declines in a process referred to as fallover [87]. Fallover 154 R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158 has been attributed to the formation of a tight binding inhibitor during catalysis [88]. However this early characterization of fallover has been complicated by the subsequent observations that (a) RuBP is oxidized to form tight binding inhibitors [89], which may also occur in vivo [90,91], and (b) the rate of fallover is increased when catalysis is conducted at higher temperatures [92], conditions also favoring RuBP oxidation. Further complicating the issue is the proposal that RuBP binding to additional ‘‘allosteric’’ sites participates in fallover and the observed reduction in Rubisco activity [93,94]. The relative importance of these multiple fallover processes in contributing to changes in Rubisco activity in vivo remains to be clearly established. It should be noted that steady state leaf photosynthesis can be successfully predicted over a wide range of conditions from knowledge of the electron transport limitations and kinetic parameters of Rubisco with respect to CO2 and O2 without knowledge of the RuBP concentration or concentrations of known effectors [95]. However, deficiencies in our understanding of Rubisco regulation sometimes become apparent if measured rates of photosynthesis are compared with estimates derived from measurements of extractable Rubisco activity and the levels of RuBP, phosphoglyceric acid, and other metabolites in the leaves [96]. A particularly striking example was obtained in comparisons of RuBP and phosphoglyceric acid levels during a diurnal light cycle using growth chamber- and field-grown plants [97]. The observed response of photosynthesis to these metabolites could be simulated in vitro by reciprocally varying the concentrations of RuBP, phosphoglyceric acid, and inorganic phosphate, but the response did not follow that predicted from the modified Michaelis– Menten Rubisco kinetic model. The problem is that Rubisco activity does not saturate at high RuBP concentrations and thus cannot be modeled by Michaelis– Menton kinetics at millimolar concentrations of this substrate [98]. The observed RuBP kinetics can be modeled assuming half-of-the-sites reactivity [99] in a dimer of the large subunits in which RuBP binding to one site increases both the Km and the Vmax for catalysis at the second site (J.C. Serviates and A.R. Portis, unpublished). Allostery between the two active sites in a dimer, and possibly between dimers, is also indicated by other observations. The binding of RuBP, the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate, and other sugar phosphates to both the carbamylated and the uncarbamylated enzyme can exhibit negative cooperativity and saturate before all eight sites are filled with a tightly bound ligand ([100–103]; E.M. Larson and A.R. Portis, unpublished). Early measurements of ligand binding to Rubisco indicated that there was only one site, identical to the active site. Subsequently, evidence for another, regula- tory, site has been reported by one research group for RuBP [104], 2-carboxyarabinitol 1,5-bisphosphate [105], inorganic phosphate [106], and 6-phosphogluconate [107]. Crystal structures of Rubisco in the presence of a variety of ligands have not provided any evidence for the location of this site. However, in one of the ‘‘open’’ Rubisco structures, inorganic phosphate was recently observed to bind outside of the active site in a ‘‘latch’’ site normally occupied by Asp-473 in the closed Rubisco structures [82]. Two widely separated residues (Lys-21 and Lys-305) appear to be indirectly involved with the allosteric site and with the occurrence of a decline in Rubisco activity when assayed in vitro [108]. These residues have also been associated with microheterogeneity among the LSs in a crystal structure of the carbamylated spinach enzyme complexed with 2carboxyarabinitol 1,5-bisphosphate [109]. However, no evidence for microheterogeneity was observed in another high-resolution structural determination of this same complex [110]. Thus, at the present time, the physiological significance of the regulatory ligand binding sites and LS structural microheterogeneity is not clear. Activase Rubisco activase appears to catalyze a rather unusual posttranslational modification of Rubisco, necessitated by the conformational changes associated with the tight binding mode of ligands such as RuBP with the uncarbamylated enzyme and 2-carboxyarabinitol 1-phosphate with the carbamylated enzyme. To facilitate the release of these tightly bound sugar phosphates and rapidly restore activity to Rubisco, plants utilize Rubisco activase to reopen the catalytic site [111]. Rubisco activase is a member of the AAAþ (ATPases associated with diverse cellular activities) protein family that constitutes a wide variety of proteins with chaperone-like functions, typically involving the disruption of molecular and macromolecular structures [112]. The three-dimensional molecular structure of activase is not known, but the AAAþ module appears to have a conserved fold [112]. Consistent with many AAAþ proteins in which the AAAþ module is linked covalently to domains that determine the actual cellular function, residues at both the N and the C termina outside of the AAAþ module in Rubisco activase appear to interact with Rubisco [113,114]. A region on the LS of Rubisco, which is adjacent to the active site, has been identified using site-directed mutagenesis to change the specificity between Chlamydomonas Rubisco and heterologous Rubisco activases [115,116]. AAAþ proteins usually participate in macromolecular complexes composed of several AAAþ protein subunits, often assembled in rings where the ATPase activity appears to act as a motor R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158 driving inter- and intramolecular movements [112]. Similarly, the activase subunits are highly self-associating, increasing in both ATPase and Rubisco activation with the extent of oligomerization [117,118]. Based on AAAþ protein analogies, the ‘‘open’’ and ‘‘closed’’ Rubisco structures, and a large body of research on the activase, a model for the mechanism of the activase and its interaction with Rubisco has been proposed [111]. Rubisco activase usually consists of two isoforms generated by alternative splicing of a pre-mRNA [119– 121]. The activity of the activase is regulated by the ADP/ATP ratio and the response is modified by redox changes in the larger isoform that are mediated by thioredoxin-f [122]. These features allow the activity of Rubisco to be regulated by the activase in response to light intensity [123] and down-regulated in response to triose phosphate use limitations [124] associated with inadequate sinks. However, some species lack a larger isoform, and the exact mode of activase/Rubisco regulation in these species remains unclear [125]. A detailed review of the activase is available [111]. Oxidation When plants are placed under various types of environmental stress, a common response is accelerated senescence and a loss of Rubisco protein. The idea that Rubisco degradation during this process is dependent upon oxidative modification of the protein emerged quite early [126] and recently has been reiterated [127,128]. However the molecular characterization of the modifications in Rubisco when plants are placed under stress has been difficult and this may be due to rapid turnover of the modified protein [129]. Several types of stresses that result in Rubisco degradation are known: UV radiation, ozone, nutrient limitations, and other stresses (e.g., osmotic or high light) that are known to generate active oxygen species (hydrogen peroxide, superoxide, and hydroxide radicals) in the chloroplasts and/or be associated with an accelerated senescence. UV radiation causes a covalent nondisulfide crosslinkage of the LS and SS (65- to 66-kDa product) attributed to Trp photolysis [130] followed by polymerization into very large molecular-mass aggregates [131,132]. The specific Trp residues involved have not been identified. The specific modifications in Rubisco associated with the exposure of intact plants to ozone are not as clear. The oxidation and aggregation of Rubisco induced by ozone exposures in vitro with the isolated enzyme or isolated chloroplasts could not be detected in intact tissue [133]. Due to its high reactivity, it is unlikely that ozone penetrates into the chloroplast and oxidizes Rubisco directly [134], but ozone can result in the forma- 155 tion of active oxygen species that could then oxidize Rubisco [133]. Carbonyl derivatives on the side chains of His, Arg, Lys, and Pro are the typical products of protein oxidation [135]. Carbonyl residues in numerous Phaseolus vulgaris proteins, including the Rubisco SS, were recently detected after chronic ozone stress [136]. Active oxygen species have also been shown to cause Rubisco breakdown into distinct fragments in isolated chloroplasts [137,138]. Active oxygen treatment of Rubisco in vitro results in modifications that allow Rubisco to become a substrate for stromal proteases [139]. The hydroxl radical has been implicated as a major player in this process [140] and Gly-329 is a key residue, which can be directly attacked by the hydroxyl radical to cause fragmentation [141]. Another residue associated with environmental stress, oxidation, and increased degradation of Rubisco is Cys [142–146]. Cys oxidation also primes Rubisco for proteolysis in vitro [143,147,148]. The formation of mixed disulfides [128] and intermolecular cross-linking of the large subunits via Cys-247 [144] may be important in Rubisco degradation in vivo. Site-directed mutagenesis in Chlamydomonas was recently used to investigate the role of Cys-172, which may form a disulfide with Cys192, in Rubisco stability. A substitution of Cys-172 with Ser resulted in an enzyme that was less stable to high temperatures in vitro, but was more resistant to protease digestion at low redox potentials [149]. However, more significantly, the stability of the enzyme in vivo to oxidative (H2 O2 ) and osmotic (mannitol) stress was increased. Site-directed mutagenesis is clearly a powerful technique to further investigate the role of specific Cys or other residues in Rubisco stability under various oxidative stresses. One of the amino acids most susceptible to oxidation is Met, which under physiological conditions leads to the formation of Met sulfoxide residues [150]. The presence of Met sulfoxide residues in polypeptides can result in functional inactivation [151,152] and targeting for proteolytic degradation [153]. However, in all organisms examined thus far, a thioredoxin-dependent enzyme, peptide Met sulfoxide reductase (PMSR), is capable of catalyzing the reduction of Met sulfoxide residues back to Met and restoration of polypeptide function [154]. A plastidal form of PMSR has been cloned from Arabidopsis and is localized in chloroplasts [155,156]. A recent report confirms the ability of chloroplastic PMSR to restore functional competency to a chloroplastic heat shock protein (Hsp21) inactivated by oxidation of Met residues [157]. An interesting and significant hypothesis with regard to the cyclic oxidation and reduction of Met residues in proteins by PMSR is that this is a cellular mechanism especially well suited to an antioxidant defense mechanism [150,158–160]. With regard to the chloroplast, the high concentration of Rubisco with 96 Met residues per holoenzyme (spinach 156 R.L. Houtz, A.R. Portis Jr. / Archives of Biochemistry and Biophysics 414 (2003) 150–158 enzyme) suggests that the continuous oxidation and reduction of methionine residues could conceivably result in a rather large consumption of oxidative metabolites. The role that cyclical oxidation and reduction of Met residues in Rubisco plays in turnover or activity remains to be elucidated. Finally, a very recent report demonstrates that nonenzymatic glycation of Rubisco by ascorbate results in increased susceptibility to proteases and suggests that glycation could play a role in determining the in vivo turnover of Rubisco [161]. Conclusions Perhaps no other enzyme so critical in the biology on this planet is subject to such extensive processing and constant conformational changes as Rubisco. In many cases these modifications and structural changes are essential for the functional role of Rubisco in photosynthetic CO2 assimilation, whereas for others the functional role(s) remain unknown. 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