Site Of Rubisco Research Articles

The activation state of Rubisco directly limits photosynthesis at low CO(2) and low O(2) partial pressures.

Using gas exchange, enzyme assays, and theoretical modeling of photosynthetic responses to light and CO(2), we investigated whether decarbamylation of the active site of Rubisco at low CO(2) and low light leads to a condition where the activation state of Rubisco directly limits the rate of net CO(2) assimilation. Photosynthetic limitation by a reduction in the activation state of Rubisco would be indicated as a decline in the initial slope of the photosynthetic CO(2) response relative to what is predicted using theoretical models. In bean (Phaseolus vulgaris) and oat (Avena sativa), we saw no discrepancy between predicted and observed initial slope values at 200 and 400 mbar O(2), indicating no limitation by the carbamylation state of Rubisco. At 30 mbar O(2) and light saturation, we also saw no discrepancy between predicted and observed initial slope values; however, at subsaturating light intensity, our observed initial slope values were less than the modeled initial slope values that corresponded to an RuBP regeneration limitation. Moreover, significant reduction of the Rubisco activation state occurred in both species at 30 mbar O(2) and 30 mubar CO(2). When the model was reprogrammed to account for observed levels of Rubisco deactivation, the predicted and measured initial slope values at low O(2) and low PPFD were similar, indicating the reduction in carbamylation state accounted for the discrepancy. We interpret this as evidence for a direct limitation of the carbamylation state of Rubisco, probably because of a CO(2) limitation for carbamate formation. This limitation was only observed at intercellular CO(2) levels below what is encountered in vivo. At physiologically relevant CO(2) levels in situ, the leaves maintained sufficient Rubisco activity to avoid cabamylation state limitations in the steady state.

Variability of the pyrenoid-based CO2 concentrating mechanism in hornworts (Anthocerotophyta).

Hornworts (Anthocerotophyta) are the only group of land plants with pyrenoid-containing chloroplasts. CO2 exchange and carbon isotope discrimination values (Δ13C) values have previously demonstrated the presence of a CO2 concentrating mechanism (CCM) in some pyrenoid-containing species. We have examined hornwort CCM function by using a combined fluorometer/mass spectrometer based technique to compare pyrenoid-containing (PhaeocerosProsk. and Notothylas Sull.) and pyrenoid-lacking (Megaceros Campbell) hornworts, with the liverwort Marchantia polymorphaL. that has standard C3 photosynthesis and a thalloid growth form similar to hornworts. We found that Notothylas has more CCM activity than Phaeoceros, and that Megaceros has the least CCM activity. Notothylas and Phaeoceros had compensation points from 11-13 parts per million (ppm) CO2, lower K0.5(CO2) than Marchantia, negligible photorespiration, and they accumulate a pool of dissolved inorganic carbon (DIC) between 19-108 nmol mg-1 chlorophyll. Megaceroshad an intermediate compensation point of 31 ppm CO2 (compared with 64 ppm CO2 in Marchantia), a lower K0.5(CO2) than Marchantia, and some photorespiration, but no DIC pool. We also determined the catalytic rate of carboxylation per active site of Rubisco for all four species (Marchantia, 2.6 s-1; Megaceros, 3.3 s-1; Phaeoceros, 4.2 s-1; Notothylas 4.3 s-1), and found that Rubisco content was 3% of soluble protein for pyrenoid-containing species, 4% for Megaceros and 8% for Marchantia.

Model of the Carbon Concentrating Mechanism in Chloroplasts of Eukaryotic Algae

A generic chloroplast-based model for the carbon concentrating mechanism (CCM) in eukaryotic algae is presented. The fine structure of chloroplasts is represented by separate compartments: marginal and bulk stroma, pyrenoid, girdle lamella, bulk thylakoids, and central lamella traversing the pyrenoid. The roles of the individual structural elements of the chloroplast with respect to the CCM and the effect of carbonic anhydrase activity in various compartments are analysed. Hypothetical HCO-3transport into the acidic thylakoid lumen is adjusted by imposing an optimization principle: a given [CO2] at the site of RuBisCO is achieved with minimum energy costs for the CCM. Our model is highly efficient in terms of saturation of RuBisCO carboxylase activity and the affinity of the chloroplast for CO2, if either a girdle lamella or a pyrenoid is present. The highest efficiency is achieved with a pyrenoid. A eukaryotic CCM is not necessarily associated with accumulation of dissolved inorganic carbon (DIC) as in cyanobacteria. Chloroplasts are categorized into four types corresponding to morphological characteristics of all major algal classes with regard to the presence of pyrenoids, girdle lamellae, and the distribution of CA activity.

Rubisco activase: an enzyme with a temperature-dependent dual function?

Heat treatment of intact spinach leaves was found to induce a unique thylakoid membrane association of an approximately 40 kDa stromal protein. This protein was identified as rubisco activase. Most of the rubisco activase was sequestered to the thylakoid membrane, particularly to the stroma-exposed regions, during the first 10 min of heat treatment at 42 degrees C. At lower temperatures (38-40 degrees C) the association of rubisco activase with the thylakoid membrane occurred more slowly. The temperature-dependent association of rubisco activase with the thylakoid membrane was due to a conformational change in the rubisco activase itself, not to heat-induced alterations in the thylakoid membrane. Association of the 41 kDa isoform of rubisco activase occurred first, followed by the binding of the 45 kDa isoform to the thylakoid membrane. Fractionation of thylakoid membranes revealed a specific association of rubisco activase with thylakoid-bound polysomes. Our results suggest a temperature-dependent dual function for rubisco activase. At optimal temperatures it functions in releasing inhibitory sugar phosphates from the active site of Rubisco. During a sudden and unexpected exposure of plants to heat stress, rubisco activase is likely to manifest a second role as a chaperone in association with thylakoid-bound ribosomes, possibly protecting, as a first aid, the thylakoid associated protein synthesis machinery against heat inactivation.

Internal conductance to CO(2) diffusion and C(18)OO discrimination in C(3) leaves.

(18)O discrimination in CO(2) stems from the oxygen exchange between (18)O-enriched water and CO(2) in the chloroplast, a process catalyzed by carbonic anhydrase (CA). A proportion of this (18)O-labeled CO(2) escapes back to the atmosphere, resulting in an effective discrimination against C(18)OO during photosynthesis (Delta(18)O). By constraining the delta(18)O of chloroplast water (delta(e)) by analysis of transpired water and the extent of CO(2)-H(2)O isotopic equilibrium (theta(eq)) by measurements of CA activity (theta(eq) = 0.75-1.0 for tobacco, soybean, and oak), we could apply measured Delta(18)O in a leaf cuvette attached to a mass spectrometer to derive the CO(2) concentration at the physical limit of CA activity, i.e. the chloroplast surface (c(cs)). From the CO(2) drawdown sequence between stomatal cavities from gas exchange (c(i)), from Delta(18)O (c(cs)), and at Rubisco sites from Delta(13)C (c(c)), the internal CO(2) conductance (g(i)) was partitioned into cell wall (g(w)) and chloroplast (g(ch)) components. The results indicated that g(ch) is variable (0.42-1.13 mol m(-2) s(-1)) and proportional to CA activity. We suggest that the influence of CA activity on the CO(2) assimilation rate should be important mainly in plants with low internal conductances.

Synthesis and characterization of magnesium and zinc complexes of 1,4,7-triazacyclononane- N-acetate. Potential models for the active site of RuBisCo

Reaction of the tetradentate pendant-arm macrocycle 1,4,7-triazacyclononane- N-acetate (L) with Zn(O 3SCF 3) 2 or Mg(O 3SCF 3) 2 in alcohol produces the six-coordinate complexes [ZnL(H 2O)]O 3SCF 3·C 2H 5OH ( 1) and [MgL(H 2O) 2]O 3SCF 3 ( 2). Compounds 1 and 2 were characterized by single-crystal X-ray crystallography. The complex cation of 1 forms a polymeric chain with zinc centers bridged in a syn, anti fashion by acetate pendant arms. The complex cation of 2 is mononuclear, with two adjacent coordination sites on magnesium occupied by water molecules. These labile coordination sites may provide a binding site for activation of substrates in functional modeling of magnesium enzymes such as RuBisCo. 1H and 13C NMR spectroscopy demonstrates that L remains bound to magnesium in methanol solution. Crystal data: 1, orthorhombic, space group P2 12 12 1, a=9.0536(7), b=14.3750(11), c=14.4741(11) Å, V=1883.7(2) Å 3, Z=4, R=0.041; 2, orthorhombic, space group P2 12 12 1, a=8.5949(3), b=13.9694(4), c=14.0076(5) Å, V=1681.83(10) Å 3, Z=4, R=0.0248.

A Theoretical Study of the Molecular Mechanism for the Carboxylation Chemistry in Rubisco

Stereochemical and structural aspects for the carbon dioxide fixation followed by hydration of d-ribulose 1,5-bisphosphate catalyzed by Rubisco are analyzed by using two model substrates, hydroxypropanone and 3,4-dihydroxy-2-pentanone. The molecular mechanisms for the carboxylation, C3-hydration and C2-inversion processes, are theoretically characterized and discussed with saddle points of index 1 representing transition structures (TS) and relevant (molded) intermediates mirroring the experimentally proposed mechanism. Ab initio SCF MO calculations at 3-21G and 6-31G** basis set levels of theory were used, while the correlation energy is included at the MP2/6-31G** level. The mapping starts from TS1, the carboxylation transition structure, which describes now the coupling of the carbon dioxide attack to the substrate C2-center in its dienol form with a synchronous interconversion of the C3 hydroxyl into a ketone group. Thereafter, water addition leads to a gem-diol followed by another step of intramolecular hydrogen transfer coupled with the C2−C3 bond breaking process. This TS breaks into one model of 3-d-phosphoglycerate product and an intermediate. The configuration inversion at the C2-center is found to be possible via intramolecular hydrogen transfer, as suggested by the corresponding TS relating the intermediate to the C2-inverted conformation. The complete set of steps found gives a self-contained description of the carboxylation followed by hydrolysis with proper stereochemistry. Comparisons between the transition state analogue: 2-carboxy-d-arabinitol-1,5-bisphosphate and TS1 show that both structures can be superposed with minimal root-mean-square deviation for C-atoms. All other calculated stationary TSs share the gross conformational features of TS1, and consequently, all molecular rearrangements detected in a vacuum can be accommodated without constraints into the active site of Rubisco. Transition structures invariances with respect to level of theory and molecular models are discussed.

Fragmentation of the Large Subunit of Ribulose-1,5-bisphosphate Carboxylase by Reactive Oxygen Species Occurs near Gly-329

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in the illuminated lysates of wheat (Triticum aestivum L.) chloroplasts is broken down by reactive oxygen radicals into 37- and 16-kDa polypeptides. Analysis of the terminal amino acid residues of both fragments revealed that the C terminus of the 37-kDa fragment was Ser-328 and the N terminus of the 16-kDa fragment was Thr-330. Gly-329, which links the two fragments, was missing, suggesting that the fragmentation of the LSU in the lysates driven by oxygen-free radicals occurs at Gly-329. Purified rubisco, exposed to a hydroxyl radical-generating system, was also cleaved at the same site of the LSU. The cleavage site was positioned at the N-terminal end of the flexible loop (loop 6) within the beta/alpha-barrel domain, constituting the catalytic site of rubisco. The binding of a reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate, to the active form of rubisco completely protected the enzyme from the fragmentation. The fragmentation was differentially affected by CO2, Mg2+, ribulose 1, 5-bisphosphate, or 2-carboxyarabinitol 1,5-bisphosphate. All these results indicate that the conformation of the catalytic site of the enzyme is involved as an important factor determining the breakdown of rubisco by reactive oxygen species. Reactive oxygen species generated at its catalytic site by a Fenton-type reaction may trigger the site-specific degradation of the LSU in the lysates of chloroplasts in the light.

Immunoblot analysis of the expression of genes for barley rubisco activase inE. coli

Rubisco activity during photosynthesis is regulated by the rubisco activase, which facilitates the dissociation of RuBP and other inhibitory sugar phosphates from the active site of rubisco in an ATP-dependent reaction. In this paper, barleyRca genes (RcaA1,RcaA2 andRcaB) were expressed inE. coli and the activity of rubisco activase expressed was assayed biochemically by chromatography. Then the protein was identified electrophoretically by SDS-PAGE and detected immunologically by Western blot analysis using polyclonal antibodies raised against the kidney bean rubisco activase as probe. The band pattern of purified proteins on the polyacrylamide gel showed two polypeptides of 46 kD and 42 kD. Anti-rubisco activase antibodies reacted specifically with both polypeptides of 46 kD and 42 kD present in the crude extracts ofE. coli transformants. Therefore, it was found that the genes of barley rubisco activase was successfully expressed inE. coli as active forms of 46 kD and 42 kD.

Enhanced CO2 alters the relationship between photosynthesis and defence in cyanogenic Eucalyptus cladocalyx F. Muell.

The effect of elevated CO2 and different levels of nitrogen on the partitioning of nitrogen between photosynthesis and a constitutive nitrogen-based secondary metabolite (the cyanogenic glycoside prunasin) was examined in Eucalyptus cladocalyx. Our hypothesis was that the expected increase in photosynthetic nitrogen-use efficiency of plants grown at elevated CO2 concentrations would lead to an effective reallocation of available nitrogen from photosynthesis to prunasin. Seedlings were grown at two concentrations of CO2 and nitrogen, and the proportion of leaf nitrogen allocated to photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), protein and prunasin compared. Up to 20% of leaf nitrogen was allocated to the cyanogenic glycoside, although this proportion varied with leaf age, position and growth conditions. Leaf prunasin concentration was strongly affected by nitrogen supply, but did not increase, on a dry weight basis, in the leaves from the elevated CO2 treatments. However, the proportion of nitrogen allocated to prunasin increased significantly, in spite of a decreasing pool of leaf nitrogen, in the plants grown at elevated concentrations of CO2. There was less protein in leaves of plants grown at elevated CO2 in both nitrogen treatments, while the concentration of active sites of Rubisco only decreased in plants from the low-nitrogen treatment. These changes in leaf chemistry may have significant implications in terms of the palatability of foliage and defence against herbivores.

Is a Low Internal Conductance to CO2 Diffusion a Consequence of Succulence in Plants with Crassulacean Acid Metabolism?

Leaf internal conductance to CO2 (gi) from substomatal cavity to the carboxylation sites of Rubisco was measured in the leaf succulent CAM species, Kalanchoe daigremontiana Hamet et Perr. Measurements were made during Rubisco-mediated atmospheric C3 carboxylation in phase IV photosynthesis. Using simultaneous gas exchange and chlorophyll fluorescence techniques, internal conductance was calculated to be 0.05 mol m-2 s-1 bar-1 , when measured at both saturating and limiting light. This is one of the lowest recorded values for gi as compared to a range of C3 species with comparable Rubisco content and indicates a large diffusion limitation to atmospheric CO2 fixation through the C3 pathway in K. daigremontiana. In ambient air, CO2 partial pressure at the carboxylation sites of Rubisco was 109 µbar. Internal diffusion is limited by a thick leaf consisting of densely packed, succulent mesophyll with a small portion of airspace. We speculate that a low internal conductance to CO2 diffusion results from the compromise between a succulent mesophyll required for C4 acid storage and access for CO2 diffusion to both PEPC in the cytoplasm and Rubisco in the chloroplasts. Restricted diffusion of CO2 within the leaf makes CO2 assimilation less efficient during the transient phases of crassulacean acid metabolism.

A detailed study of the active site of Rubisco by X-ray crystallography

A detailed study of the active site of Rubisco by X-ray crystallography

Different location in dark-adapted leaves of Phaseolus vulgaris of ribulose-1,5-bisphosphate carboxylase/oxygenase and 2-carboxyarabinitol 1-phosphate

In situ RuBisCO activity was analyzed in order to determine whether 2-carboxyarabinitol 1-phosphate (CA1P) interacts with the enzyme in dark-adapted leaves of Phaseolus vulgaris. Leaves ground to fine powder in liquid nitrogen were put directly into a reaction mixture containing a saturating concentration of ribulose 1,5-bisphosphate (RuBP) to preserve the activity of RuBisCO which was in the chloroplasts. Some 70% of the total catalytic sites of RuBisCO possessed carboxylase activity in this assay, however, RuBisCO was inhibited in the absence of RuBP. CA1P seemed to be concentrated in the veins. These results indicate that RuBisCO was not complexed with CA1P in leaves.

Inactivation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase during Catalysis. A Theoretical Study of Related Transition Structures

Possible mechanistic paths for self-inhibition of rubisco have been theoretically characterized by using analytical gradients with both AM1 semiempirical and HF/3-21G level calculations. Starting from the framework of an enediol moiety previously obtained from characterizations of the saddle point of index 1 (SPi-1) for the carboxylation and oxygenation reactions, the formation of xylulose, 3-ketoribitol, and 3-ketoarabinitol inhibitors is made possible by following specific intramolecular hydrogen rearrangements. The xylulose is attained from the SPi-1 describing intramolecular enolization via an intermediate made by protonation of the hydroxyl group linked to the third carbon (C3) of the model substrate 3,4-dihydroxy-2-pentanone. One of these two hydrogens can migrate toward C3 with the correct stereochemistry to form xylulose inhibitor. 3-Ketoribitol and 3-ketoarabinitol inhibitors can be obtained after the enediol moiety is formed. Another minimum energy structure is found which is derived from the enediol via a SPi-1 corresponding to a retroenolization. This process finishes by forming a protonated hydroxyl group at C2. From this, and following the SPi-1, the 3-ketoribitol and the 3-ketoarabinitol inhibitors can be formed. The carbon and oxygen frameworks of the stationary geometries characterized in vacuo fit well at the active site of rubisco, except perhaps for xylulose inhibitor. This geometric overlap with an experimentally determined transition state analog suggests that the active site can accommodate the interconversion chemistry found with the present approach which leads to self-inhibition. This is compatible with the hypothesis that the loss of activity is due to the products of substrate isomerization formed during catalysis.

Rubisco: the consequences of altering its expression and activation in transgenic plants

Abstract Transgenic tobacco (Nicotiana tabacum W38) hemizygous for a single antisense gene directed against Rubisco's small subunit had 35% of the Rubisco content of control leaves (15% when homozygous). CO2 assimilation (at 1000 μmol quanta m−2 s−1 and 350 μbar CO2) by the hemizygous leaves was reduced to 40% of that of the controls without material effect on stomatal conductance, chlorophyll content or other photosynthetic components. Leaf soluble protein was reduced commensurately with the reduction in Rubisco. CO2 assimilation rate in the hemizygous leaves remained limited by Rubisco activity at all, even very high, CO2 concentrations. This led to a simple, hyperbolic response of photosynthesis to intraplastid CO2 concentration from which the in vivo catalytic properties of Rubisco were inferred and compared with those of isolated Rubisco in vitro. Using a similar approach, the content of Rubisco activase was suppressed by incorporating a partial cDNA for activase into the tobacco genome in the antisense orientation with respect to a cauliflower mosaic virus 35S promoter. The progeny of a primary transformant with two anti-activase inserts had from < 1% to 20% of the activase content of control plants. Quite severe suppression of activase, to less than 5% of the amount present in control leaves, was required before effects on photosynthesis and growth became apparent, indicating that one activase tetramer must be able to service, continuously, as many as 200 Rubisco octamers. Plants with lower activase contents could not grow unless the atmosphere was enriched with CO2. Their Rubisco was less carbamylated and they had lower CO2 assimilation rates than the controls. The rate of release of 2′-carboxyarabinitol-1-phosphate from Rubisco after illumination of the antiactivase leaves was also impaired. Older anti-activase plants accumulated increasing amounts of Rubisco in their younger leaves, but were unable to carbamylate it. The photosynthetic rate per carbamylated Rubisco active site in the strongly suppressed anti-activase leaves was only approximately 25% of that seen in control leaves, suggesting that activase may not only promote carbamytation of uncarbamylated Rubisco sites, but also accelerate turnover at carbamylated sites.

Regulation of Photosynthesis in C3 and C4 Plants: A Molecular Approach.

Most plants use the C3 pathway of photosynthesis, also called the photosynthetic carbon reduction cycle (PCR), shown in Figure 1A. C3 plants have a single chloroplast type that performs all of the reactions that convert light energy into the chemical energy that is used to fix COp and to synthesize the reduced carbon compounds upon which all life depends. Ribulose-i ,5-bisphosphate carboxylaseloxygenase (Rubisco) catalyzes primary carbon fixation, in which a fivecarbon sugar phosphate, ribulose-l,5-bisphosphate (RuBP), and COp are converted to two molecules of the threecarbon compound 3-phosphoglycerate (hence the name C3). Phosphoglycerate is then phosphorylated and reduced by the products of the light reactions of photosynthesis (ATP and NADPH) to produce triose phosphate (TP). TP can be exported from the chloroplast via the chloroplast envelope phosphate (Pi) transporter to the cytosol and used in the synthesis of sucrose, which is then translocated throughout the plant (see Sonnewald et al., 1994), or it can be retained within the chloroplast for starch synthesis or recycling to RuBP. Rubisco also catalyzes the fixation of Op in a process known as photorespiration, which competes directly with fixation of COp. At air levels of Coa, for every three COp molecules fixed by Rubisco to form 3-phosphoglycerate, approximately one O2 molecule is fixed, producing Sphosphoglycerate and 3-phosphoglycolate (Figure 1A). Because 3-phosphoglycolate cannot be used in the PCR cycle, it must be recycled to phosphoglycerate via the photorespiratory pathway, expending ATP and NADPH. This competition between 0 2 and COp and the energy costs associated with recycling phosphoglycolate largely determine the efficiency of C3 photosynthesis in air (Hatch, 1988; Woodrow and Berry, 1988). The C4 pathway is a complexadaptation of the C3 pathway that overcomes the limitation of photorespiration and is found in a diverse collection of species, many of which grow in hot climates with sporadic rainfall. The C4 pathway effectively suppresses photorespiration by elevating the C02 concentration at the site of Rubisco using a biochemical C02 pump. C4 plants have two chloroplast types, each found in a specialized cell type. Leaves of C4 plants show extensive vascularization,

A signature of the oxygenase intermediate in catalysis by ribulose-bisphosphate carboxylase/oxygenase as provided by a site-directed mutant.

An uncharacterized minor transient product, observed in our earlier studies of substrate turnover by the E48Q mutant of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase (Lee, E. H., Harpel, M. R., Chen, Y.-R., and Hartman, F. C. (1993) J. Biol. Chem. 268, 26583-26591), becomes a major product when it is trapped and stabilized with borate as an additive to the reaction mixture. Chemical characterization establishes this novel product as D-glycero-2,3-pentodiulose 1,5-bisphosphate, thereby demonstrating oxidation of the C-3 hydroxyl of D-ribulose 1,5-bisphosphate to a carbonyl. As the formation of the novel oxidation product is oxygen-dependent and generates hydrogen peroxide, its precursor must be a peroxy derivative of ribulose bisphosphate. Thus, discovery of the dicarbonyl bisphosphate lends direct support to the long standing, but heretofore unproven, postulate that the normal pathway for oxidative cleavage of ribulose bisphosphate by the wild-type enzyme entails a peroxy intermediate. Our results also suggest that stabilization of the peroxy intermediate by the wild-type enzyme promotes carbon-carbon scission as opposed to elimination of hydrogen peroxide.

Limitation of net CO2 assimilation rate by internal resistances to CO2 transfer in the leaves of two tree species (Fagus sylvatica L. and Castanea sativa Mill.)

ABSTRACTUsing a combination of gas‐exchange and chlorophyll fluorescence measurements, low apparent CO2/O2 specificity factors (1300 mol mol−1) were estimated for the leaves of two deciduous tree species (Fagus sylvatica and Castanea sativa). These low values contrasted with those estimated for two herbaceous species and were ascribed to a drop in the CO2 mole fraction between the intercellular airspace (Ci) and the catalytic site of Rubisco (Cc) due to internal resistances to CO2 transfer. Cc. was calculated assuming a specificity of Rubisco value of 2560 mol mol−1. The drop between Ci and Cc was used to calculate the internal conductance for CO2 (gi). A good correlation between mean values of net CO2 assimilation rate (A) and gi was observed within a set of data obtained using 13 woody plant species, including our own data. We report that the relative limitation of A, which can be ascribed to internal resistances to CO2 transfer, was 24–30%. High internal resistances to CO2 transfer may explain the low apparent maximal rates of carboxylation and electron transport of some woody plant species calculated from A/Ci curves.

Evidence That 2-Carboxyarabinitol 1-Phosphate Binds to Ribulose-1,5-Bisphosphate Carboxylase in Vivo.

An important question concerning the role of carboxyarabinitol 1-phosphate (CA1P) metabolism in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity is the extent to which CA1P is bound to Rubisco in vivo. We report here the development of an extraction procedure using ammonium sulfate that stabilizes CA1P bound to Rubisco. This procedure exploits the ability of sulfate to bind at the catalytic site of Rubisco and to competitively balance the binding and release of CA1P from Rubisco. In darkened bean leaves about 75% of the Rubisco catalytic sites were found to be bound with CA1P. This confirms previous indirect estimates from gas exchange measurements. We have used this extraction procedure to examine CA1P-Rubisco interactions in bean during a natural transition from darkness to light. With increasing light intensity following sunrise, CA1P degradation proceeded in two distinct phases: first, a majority of the unbound CA1P pool was degraded at very low light levels ([less than or equal to]30 [mu]mol quanta m-2 s-1); second, CA1P initially bound to Rubisco was then degraded at increasing light levels (>30 [mu]mol quanta m-2 s-1). These results indicate that there is a low-fluence activation of CA1P phosphatase that can occur prior to CA1P release by Rubisco activase. This activation may be mediated by NADPH. During sunrise in bean, the level of the catalytically competent form of Rubisco was regulated by CA1P metabolism.

Photoaffinity labeling of ribulose-1,5-bisphosphate carboxylase/oxygenase activase with ATP gamma-benzophenone. Identification of the ATP gamma-phosphate binding domain

The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photo-affinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]ATP gamma BP). Covalent incorporation of [gamma-32P]ATP gamma BP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabelling of Rubisco activase with ATP gamma BP exhibited saturation kinetics; the apparent Kd for photolabeling was 5 microM. Two lines of evidence showed that ATP gamma BP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATP gamma BP. Second, photolysis of Rubisco activase in the presence of ATP gamma BP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATP gamma BP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATP gamma BP was identified by isolating photolabeled peptides. Sequence analysis showed that ATP gamma BP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP gamma-phosphate-binding domain of Rubisco activase with ATP gamma BP.