Experimental evidence for extra proton exchange in ribulose 1,5-bisphosphate carboxylase/oxygenase catalysis.

Despite considerable advances in the past 50 y, the mechanism of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalysis is still not well understood. In particular, the movement and exchange of protons within the active site is not well documented: typically, kinetics of H exchange during the first steps of catalysis, i.e. abstraction of the H3 atom of ribulose 1,5-bisphosphate (RuBP) and enolization, are not clearly established. Here, we took advantage of reaction assays run in heavy water (2H2O) to monitor the appearance of deuterated RuBP and deuterated products (3-phosphoglycerate and 2-phosphoglycolate) with exact mass LC-MS. Enolization was reversible such that de-enolization generated not only monodeuterated RuBP (2H-[H-3]-ribulose 1,5-bisphosphate) but also dideuterated RuBP (2H2-[H-3,O-3]-ribulose 1,5-bisphosphate). Carboxylation yielded about one half deuterated 3-phosphoglycerate (2H-[H-2]-3-phosphoglycerate) and also a small proportion of dideuterated 3-phosphoglycerate (2H2-[H-2,O-2]-3-phosphoglycerate). Oxygenation generated a small amount of monodeuterated, but no dideuterated, products. (Di)deuterated isotopologue abundance depended negatively on gas concentration. We conclude that in addition to the first step of proton exchange at H3 occurring before gas addition (and thus influenced by the competition between de-enolization and gas addition), there is another proton exchange step between solvent water, active site residues, and the 2,3-enediol(ate) leading to deuterated OH groups in products.

Ribulose 1,5-bisphosphate carboxylase/oxygenase activates O2 by electron transfer.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the cornerstone of atmospheric CO2 fixation by the biosphere. It catalyzes the addition of CO2 onto enolized ribulose 1,5-bisphosphate (RuBP), producing 3-phosphoglycerate which is then converted to sugars. The major problem of this reaction is competitive O2 addition, which forms a phosphorylated product (2-phosphoglycolate) that must be recycled by a series of biochemical reactions (photorespiratory metabolism). However, the way the enzyme activates O2 is still unknown. Here, we used isotope effects (with 2H, 25Mg, and 18O) to monitor O2 activation and assess the influence of outer sphere atoms, in two Rubisco forms of contrasted O2/CO2 selectivity. Neither the Rubisco form nor the use of solvent D2O and deuterated RuBP changed the 16O/18O isotope effect of O2 addition, in clear contrast with the 12C/13C isotope effect of CO2 addition. Furthermore, substitution of light magnesium (24Mg) by heavy, nuclear magnetic 25Mg had no effect on O2 addition. Therefore, outer sphere protons have no influence on the reaction and direct radical chemistry (intersystem crossing with triplet O2) does not seem to be involved in O2 activation. Computations indicate that the reduction potential of enolized RuBP (near 0.49 V) is compatible with superoxide (O2•-) production, must be insensitive to deuteration, and yields a predicted 16O/18O isotope effect and energy barrier close to observed values. Overall, O2 undergoes single electron transfer to form short-lived superoxide, which then recombines to form a peroxide intermediate.

Rubisco is not really so bad.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most widespread carboxylating enzyme in autotrophic organisms. Its kinetic and structural properties have been intensively studied for more than half a century. Yet important aspects of the catalytic mechanism remain poorly understood, especially the oxygenase reaction. Because of its relatively modest turnover rate (a few catalytic events per second) and the competitive inhibition by oxygen, Rubisco is often viewed as an inefficient catalyst for CO2 fixation. Considerable efforts have been devoted to improving its catalytic efficiency, so far without success. In this review, we re-examine Rubisco's catalytic performance by comparison with other chemically related enzymes. We find that Rubisco is not especially slow. Furthermore, considering both the nature and the complexity of the chemical reaction, its kinetic properties are unremarkable. Although not unique to Rubisco, oxygenation is not systematically observed in enolate and enamine forming enzymes and cannot be considered as an inevitable consequence of the mechanism. It is more likely the result of a compromise between chemical and metabolic imperatives. We argue that a better description of Rubisco mechanism is still required to better understand the link between CO2 and O2 reactivity and the rationale of Rubisco diversification and evolution.

Carbon allocation to major metabolites in illuminated leaves is not just proportional to photosynthesis when gaseous conditions (CO2 and O2 ) vary.

In gas-exchange experiments, manipulating CO2 and O2 is commonly used to change the balance between carboxylation and oxygenation. Downstream metabolism (utilization of photosynthetic and photorespiratory products) may also be affected by gaseous conditions but this is not well documented. Here, we took advantage of sunflower as a model species, which accumulates chlorogenate in addition to sugars and amino acids (glutamate, alanine, glycine and serine). We performed isotopic labelling with 13 CO2 under different CO2 /O2 conditions, and determined 13 C contents to compute 13 C-allocation patterns and build-up rates. The 13 C content in major metabolites was not found to be a constant proportion of net fixed carbon but, rather, changed dramatically with CO2 and O2 . Alanine typically accumulated at low O2 (hypoxic response) while photorespiratory intermediates accumulated under ambient conditions and at high photorespiration, glycerate accumulation exceeding serine and glycine build-up. Chlorogenate synthesis was relatively more important under normal conditions and at high CO2 and its synthesis was driven by phosphoenolpyruvate de novo synthesis. These findings demonstrate that carbon allocation to metabolites other than photosynthetic end products is affected by gaseous conditions and therefore the photosynthetic yield of net nitrogen assimilation varies, being minimal at high CO2 and maximal at high O2 .

D2O solvent isotope effects suggest uniform energy barriers in ribulose-1,5-bisphosphate carboxylase/oxygenase catalysis.

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme on Earth and is responsible for the fixation of atmospheric CO(2) into biomass. The reaction consists of incorporation of CO(2) and solvent H(2)O into d-ribulose 1,5-bisphosphate (RuBP) to yield 3-phospho-d-glycerate. The reaction involves several proton-dependent events: abstraction and protonation during enolization of RuBP and hydrolysis and reprotonation of the six-carbon reaction intermediate (carboxyketone). Although much is known about Rubisco structure and diversity, fundamental aspects of the reaction mechanism are poorly documented. How and when are protons exchanged among substrate, amino acid residues, and solvent water, and could alterations of proton exchange influence catalytic turnover? What is the energy profile of the reaction? To answer these questions, we measured catalytic rates and the (12)CO(2)/(13)CO(2) isotope effect in isotopic waters. We show that with increasing D(2)O content, the maximal carboxylation velocity (k(cat)(c)) decreased linearly and was 1.7 times lower in pure D(2)O. By contrast, the isotope effect on the apparent Michaelis constant for CO(2) (K(c)) was unity, suggesting that H/D exchange might have occurred with the solvent in early steps thereby slowing the overall catalysis. Calculations of kinetic commitments from observed isotope effects further indicate that (1) enolization and processing of the carboxyketone are similarly rate-limiting and (2) the tendency of the carboxyketone to go backward (decarboxylation) is likely exacerbated upon deuteration. Our results thus suggest that Rubisco catalysis is achieved by a rather equal distribution of energy barriers along the reaction.

On the resilience of nitrogen assimilation by intact roots under starvation, as revealed by isotopic and metabolomic techniques.

The response of root metabolism to variations in carbon source availability is critical for whole-plant nitrogen (N) assimilation and growth. However, the effect of changes in the carbohydrate input to intact roots is currently not well understood and, for example, both smaller and larger values of root:shoot ratios or root N uptake have been observed so far under elevated CO(2). In addition, previous studies on sugar starvation mainly focused on senescent or excised organs while an increasing body of data suggests that intact roots may behave differently with, for example, little protein remobilization. Here, we investigated the carbon and nitrogen primary metabolism in intact roots of French bean (Phaseolus vulgaris L.) plants maintained under continuous darkness for 4 days. We combined natural isotopic (15)N/(14)N measurements, metabolomic and (13)C-labeling data and show that intact roots continued nitrate assimilation to glutamate for at least 3 days while the respiration rate decreased. The activity of the tricarboxylic acid cycle diminished so that glutamate synthesis was sustained by the anaplerotic phosphoenolpyruvate carboxylase fixation. Presumably, the pentose phosphate pathway contributed to provide reducing power for nitrate reduction. All the biosynthetic metabolic fluxes were nevertheless down-regulated and, consequently, the concentration of all amino acids decreased. This is the case of asparagine, strongly suggesting that, as opposed to excised root tips, protein remobilization in intact roots remained very low for 3 days in spite of the restriction of respiratory substrates.

Is there any 12C/13C fractionation during starch remobilisation and sucrose export in potato tubers?

The delta(13)C (carbon isotope composition) variations in respired CO(2), total organic matter, proteins, sucrose and starch have been measured during tuber sprouting of potato (Solanum tuberosum) in darkness. Measurements were carried out both on tubers and on their growing sprouts for 23 days after the start of sprout development. Sucrose was slightly (13)C-depleted compared with starch in tubers, suggesting that starch breakdown was associated with a small isotope fractionation. In sprouts, all biochemical fractions including sucrose were (13)C-enriched compared with source tuber-sucrose, suggesting that sucrose translocation from tuber to sprouts fractionated against (12)C. However, both apparent fractionations were explained by the consumption of (13)C-depleted carbon for respiration or growth that enriched in the (13)C sucrose molecules left behind. In addition, whole tuber sucrose is constantly composed of recent sucrose from starch breakdown and old sucrose associated with an inherited, slightly (13)C-depleted pool. We therefore conclude that any fractionation at either the starch breakdown or the sucrose translocation level is unlikely under our conditions.

Carbon stable isotope ratio of phloem sugars in mature pine trees throughout the growing season: comparison of two extraction methods.

The study presents a comparison of two phloem sugar extraction methods. The amount of phloem sugar extracted and the carbon isotope composition (delta(13)C) of the total extracts and of the main phloem compounds separated by high-performance liquid chromatography (sucrose, glucose, fructose and pinitol) are compared. These two phloem sap extraction methods are exudation in distilled water and a new method using centrifugation, which avoids the addition of any solvent. We applied both extraction methods on phloem discs sampled from 38-year-old Pinus pinaster trees in south-western France throughout the period from June 2007 to December 2008 on different time-scales: hourly, daily and monthly. We found that the centrifugation method systematically extracted ca. 50% less compounds from the phloem discs than the exudation method. In addition, the two extraction methods provided similar delta(13)C values of the total extracts, but the values obtained by the exudation method were 0.6 per thousand more negative than those calculated from the mass balance using the individual constituents. Over the growing season, both extraction methods exhibited lower total sugar content and more (13)C-enriched phloem sap in summer compared with winter values. These findings suggest that both extraction methods can be applied to study the carbon isotope composition of phloem sap, and the centrifugation method has the advantage that no solvent has to be added. The exudation method, however, is more appropriate for the quantification of the amounts of phloem sugars.

Kinetic 12C/13C isotope fractionation by invertase: evidence for a small in vitro isotope effect and comparison of two techniques for the isotopic analysis of carbohydrates.

The natural (13)C/(12)C isotope composition (delta(13)C) of plants and organic compounds within plant organs is a powerful tool to understand carbon allocation patterns and the regulation of photosynthetic or respiratory metabolism. However, many enzymatic fractionations are currently unknown, thus impeding our understanding of carbon trafficking pathways within plant cells. One of them is the (12)C/(13)C isotope effect associated with invertases (EC 3.2.1.26) that are cornerstone enzymes for Suc metabolism and translocation in plants. Another conundrum of isotopic plant biology is the need to measure accurately the specific delta(13)C of individual carbohydrates. Here, we examined two complementary methods for measuring the delta(13)C value of sucrose, glucose and fructose, that is, off-line high-performance liquid chromatography (HPLC) purification followed by elemental analysis and isotope ratio mass spectrometry (EA-IRMS) analysis, and gas chromatography-combustion (GC-C)-IRMS. We also used these methods to determine the in vitro (12)C/(13)C isotope effect associated with the yeast invertase. Our results show that, although providing more variable values than HPLC approximately EA-IRMS, and being sensitive to derivatization conditions, the GC-C-IRMS method gives reliable results. When applied to the invertase reaction, both methods indicate that the (12)C/(13)C isotope effect is rather small and it is not affected by the use of heavy water (D(2)O).

Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods.

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO(2) fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO(2) flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C(4) leaf spiked with C(3) starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems.

Metabolic origin of the delta13C of respired CO2 in roots of Phaseolus vulgaris.

Root respiration is a major contributor to soil CO2 efflux, and thus an important component of ecosystem respiration. But its metabolic origin, in relation to the carbon isotope composition (delta13C), remains poorly understood. Here, 13C analysis was conducted on CO2 and metabolites under typical conditions or under continuous darkness in French bean (Phaseolus vulgaris) roots. 13C contents were measured either under natural abundance or following pulse-chase labeling with 13C-enriched glucose or pyruvate, using isotope ratio mass spectrometer (IRMS) and nuclear magnetic resonance (NMR) techniques. In contrast to leaves, no relationship was found between the respiratory quotient and the delta13C of respired CO2, which stayed constant at a low value (c. -27.5 per thousand) under continuous darkness. With labeling experiments, it is shown that such a pattern is explained by the 13C-depleting effect of the pentose phosphate pathway; and the involvement of the Krebs cycle fueled by either the glycolytic input or the lipid/protein recycling. The anaplerotic phosphoenolpyruvate carboxylase (PEPc) activity sustained glutamic acid (Glu) synthesis, with no net effect on respired CO2. These results indicate that the root delta13C signal does not depend on the availability of root respiratory substrates and it is thus plausible that, unless the 13C photosynthetic fractionation varies at the leaf level, the root delta13C signal hardly changes under a range of natural environmental conditions.

Biotic and abiotic factors affecting the delta(13)C of soil respired CO(2) in a Mediterranean oak woodland.

The flux (R(s)) and carbon isotopic composition (delta(13)C (Rs)) of soil respired CO (2) was measured every 2 h over the course of three diel cycles in a Mediterranean oak woodland, together with measurements of the delta(13)C composition of leaf, root and soil organic matter (delta(13)C (SOM)) and metabolites. Simulations of R(s) and delta(13)C (Rs) were also made using a numerical model parameterised with the SOM data and assuming short-term production rates were driven mainly by temperature. Average values of delta(13)C (Rs) over the study period were within the range of root metabolite and average delta(13)C (SOM) values, but enriched in (13)C relative to the bulk delta(13)C of leaf, litter, and roots and the upper soil organic layers. There was good agreement between model output and observed CO (2) fluxes and the underlying features of delta(13)C (Rs). Observed diel variations of 0.5 per thousand in delta(13)C (Rs) were predicted by the model in response to temperature-related shifts in production rates along a approximately 3 per thousand gradient observed in the profile of delta(13)C (SOM). However, observed delta(13)C (Rs) varied by over 2 per thousand, indicating that both dynamics in soil respiratory metabolism and physical processes can influence short-term variability of delta(13)C (Rs).

Divergence in delta(13)C of dark respired CO(2) and bulk organic matter occurs during the transition between heterotrophy and autotrophy in Phaseolus vulgaris plants.

Substantial evidence has been published in recent years demonstrating that postphotosynthetic fractionations occur in plants, leading to (13)C-enrichment in heterotrophic (as compared with autotrophic) organs. However, less is known about the mechanism responsible for changes in these responses during plant development. The isotopic signature of both organic matter and respired CO(2) for different organs of French bean (Phaseolus vulgaris) was investigated during early ontogeny, in order to identify the developmental stage at which isotopic changes occur. Isotopic analyses of metabolites and mass balance calculations helped to constrain the metabolic processes involved. At the plant scale, apparent respiratory fractionation was constantly positive in the heterotrophic phase (c. 1 per thousand) and turned negative with autotrophy acquisition (down to -3.08 per thousand). Initially very close to that of the dry seed (-26.83 +/- 0.69 per thousand), isotopic signatures of organic matter and respired CO(2) diverged (in opposite directions) in leaves and roots after onset of photosynthesis. Respired CO(2) reached values up to -20 per thousand in leaves and became (13)C-depleted down to -29 per thousand in roots. It was concluded that isotopic differences between organs occurred subsequent to metabolic changes in the seedling during the transition from heterotrophy to autotrophy. They were especially related to respiration and respiratory fractionation.