A sensitive,simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O2 specificity factor

A simple approach to determine CO₂/O₂ specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO₂ fixation at varying [CO₂]/[O₂] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of ¹⁴CO₂ incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO₂]/[O₂] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO₂/O₂ specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO₂]/[O₂] ratios examined, ranging from 14 to 215 micromolar CO₂ (provided as 1–16 mM NaHCO₃) and 614 micromolar O₂ provided as 50% O₂. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid) - L large subunit of rubisco - PGA 3-phosphoglyceric acid - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - S small subunit of rubisco - XuBP d-xylulose 1,5-bisphosphate     

Effects of chronic ozone and elevated atmospheric CO2 concentrations on ribulose-1,5-bisphosphate in soybean (Glycine max)

Ribulose-1,5-bisphosphate (RuBP) pool size was determined at regular intervals during the growing season to understand the effects of tropospheric ozone concentrations, elevated atmospheric carbon dioxide concentrations and their interactions on the photosynthetic limitation by RuBP regeneration. Soybean (Glycine max [L.] Merr. cv. Essex) was grown from seed to maturity in open-top field chambers in charcoal-filtered air (CF) either without (22 nmol O₃ mol^?1) or with added O₃ (83 nmol mol^?1) at ambient (AA, 369 μmol CO₂ mol^?1) or elevated CO₂ (710 μmol mol^?1). The RuBP pool size generally declined with plant age in all treatments when expressed on a unit leaf area and in all treatments but CF-AA when expressed per unit ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) binding site. Although O₃ in ambient CO₂ generally reduced the RuBP pool per unit leaf area, it did not change the RuBP pool per unit Rubisco binding site. Elevated CO₂, in CF or O₃-fumigated air, generally had no significant effect on RuBP pool size, thus mitigating the negative O₃ effect. The RuBP pools were below 2 mol mol^?1 binding site in all treatments for most of the season, indicating limiting RuBP regeneration capacity. These low RuBP pools resulted in increased RuBP regeneration via faster RuBP turnover, but only in CF air and during vegetative and flowering stages at elevated CO₂. Also, the low RuBP pool sizes did not always reflect RuBP consumption rates or the RuBP regeneration limitation relative to potential carboxylation (%RuBP). Rather, %RuBP increased linearly with decrease in the RuBP pool turnover time. These data suggest that amelioration of damage from O₃ by elevated atmospheric CO₂ to the RuBP regeneration may be in response to changes in the Rubisco carboxylation.     

Specificity factor of Rubisco: estimation in intact leaves by carboxylation at different CO2/O2 ratios

The specificity factor of Rubisco (S _f) was estimated in intact leaves from the carboxylation of ribulose-1,5-bisphosphate (RuBP) at various CO₂/O₂ ratios. As oxygenation is calculated by the difference of the ¹⁴CO₂ uptake by RuBP in the absence and presence of oxygen, it is important to choose the optimum CO₂/O₂ ratios. At high CO₂ concentration (1,000 cm³ m^?3 and higher) oxygenation consumes less than 50% RuBP but the difference of concentrations of CO₂ at cell walls (C _w) and at the carboxylation centers (C _c) is 2?C5% and the influence of mesophyll resistance (r _md) is of minor importance. To accumulate large endogenous pool of RuBP, the leaves were preilluminated in the CO₂- and O₂-free gas environments for 8 to 10 s. Thereafter the light was switched off and the leaves were flushed with the gas containing different concentrations of ¹⁴CO₂ and O₂. The specificity factor of Rubisco was calculated from the amount of the tracer taken up under different ¹⁴CO₂/O₂ ratios by the exhaustion of the RuBP pool. Application of ¹⁴CO₂ allowed us to discriminate between the CO₂ uptake and the concurrent respiratory CO₂ release which proceeded at the expense of unlabelled intermediates.     

Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2)

Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO₂ to activate the enzyme, changes in CO₂ between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO₂ levels and 21% O₂ or 1% or less O₂, the levels of ribulose bisphosphate were high and not limiting for CO₂ fixation. With high leaf ribulose bisphosphate, the K_act(CO₂) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO₂ and O₂, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.

The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg²⁺ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg²⁺ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO₂-Mg²⁺-RuBP forms. Higher irradiances favor more optimal Mg²⁺ and pH, with greater activation of the carboxylase and increased photosynthesis.

     

Activation of Rubisco controls CO<Subscript>2</Subscript> assimilation in light: a perspective on its discovery

CO₂ fixation during photosynthesis is regulated by the activity of ribulose bisphosphate carboxylase (Rubisco). This conclusion became more apparent to me after CO₂-fixation experiments using isolated spinach chloroplasts and protoplasts, purified Rubisco enzyme, and intact leaves. Ribulose bisphosphate (RuBP) pools and activation of Rubisco were measured and compared to ¹⁴CO₂ fixation in light. The rates of ¹⁴CO ₂ assimilation best followed the changes in Rubisco activation under moderate to high light intensities. RuBP pool sizes regulated ¹⁴ ₂ assimilation only in very high CO₂ levels, low light and in darkness. Activation of Rubisco involves two separate processes: carbamylation of the protein and removal of inhibitors blocking carbamylation or blocking RuBP binding to carbamylated sites before reaction with CO₂ or O₂. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Sensitive Assay Procedure for Simultaneous Determination of Ribulose-1,5-bisphosphate Carboxylase and Oxygenase Activities

A sensitive assay procedure is described for the simultaneous determination of ribulose-1,5-bisphosphate (RuBP) carboxylase and oxygenase activities. In this assay, [1-³H]RuBP is incubated with ¹⁴CO₂ and O₂. Carboxylation rate is determined from ¹⁴CO₂ incorporation and oxygenation rate is determined from [2-³H]glycolate-phosphate production. The assay was found to be suitable at all CO₂ and O₂ concentrations examined, which ranged from 0 to 300 micromolar CO₂ (20 millimolar NaHCO₃) and 0 to 1.15 millimolar (100%) O₂. In combination with a polarographic assay, the stoichiometry of the RuBP oxygenase reaction was found to be RuBP-O₂-glycolate phosphate-glycerate phosphate (1:1:1:1).     

Enzymic Fractionation of the Stable Carbon Isotopes of Carbon Dioxide by Ribulose-1,5-bisphosphate Carboxylase

The enzymic fractionation of the stable carbon isotopes of CO₂ (Δco₂) was determined using a purified preparation of ribulose-1,5-bisphosphate (RuBP) carboxylase isolated from cotton (a C₃ plant) leaves. The bicarbonate concentration in the reaction mixture saturated the enzyme and furnished an infinite pool of ¹²CO₂ and ¹³CO₂ for enzyme fractionation. The RuBP was 96 to 98% pure. The phosphoglycerate synthesized in the reaction mixtures was purified free of RuBP, phosphoglycolate, and other phosphate esters by column chromatography on Dowex 1-Cl⁻ resin. The average Δco₂ value of −27.1% was determined from five separate experiments. A discussion of the isotope fractionation associated with photosynthetic CO₂ fixation in plants shows that the enzymic fractionation of stable carbon isotopes of CO₂ by RuBP carboxylase is of major importance in determining the δ¹³C values of C₃ plants.     

Simple Determination of the CO(2)/O(2) Specificity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by the Specific Radioactivity of [C]Glycerate 3-Phosphate

A new method is presented for measurement of the CO₂/O₂ specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [¹⁴C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. ¹⁴CO₂ fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO₂ in O₂-saturated water and carboxylase only with 160 micromolar CO₂ under N₂. Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [¹⁴C]PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 ± 4), from the green alga Chlamydomonas reinhardtii (66 ± 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.     

How various factors influence the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO₂/O₂ specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO₂/O₂ specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O₂ has a higher free energy of activation than the corresponding reaction of this substrate with CO₂. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO₂/O₂ specificity that is observed when activator Mg²⁺ is replaced by Mn²⁺ may be due to Mg²⁺ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn²⁺ is a transition-metal ion that can overcome the triplet character of O₂ to promote the oxygenation reaction.Abbreviations CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K_c K_mfor CO₂ - K_o K_mfor O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V_c V _max for carboxylation - V_o V _max for oxygenation     

Photosynthesis and Activity of Ribulose Bisphosphate Carboxylase of Wheat and Maize Seedlings during and following Exposure to O(2)-Low, CO(2)-Free N(2)

Seven day old wheat and maize seedlings were exposed to 1300 or 2000 microeinsteins per square meter per second photosynthetically active radiation in CO₂-free air for 3 hours with either 1% O₂ in N₂ or N₂-only and then returned to normal air of 340 microliters per liter CO₂, 21% O₂ in N₂. Activity of the ribulose bisphosphate carboxylase and amount of the substrate, ribulose 1,5-bisphosphate, were measured during and following the CO₂-free treatments as was photosynthetic CO₂ fixation. Photoinhibition of photosynthesis was observed only with wheat seedlings following the N₂ only treatment. During the CO₂-free treatments, the levels of RuBP rose during all experiments except when wheat was photoinhibited. The activity of the ribulose bisphophate carboxylase, measured directly upon grinding the leaves, declined during the CO₂-free conditions. The carboxylase total activity increased in minutes in the leaf during and following the CO₂-free treatments. The specific activities of the wheat carboxylase went from 0.16 to 1.06 micromoles CO₂ fixed per milligram protein per minute while the maize carboxylase varied from 0.05 to 0.36 micromole CO₂ fixed per millogram protein per minute. This suggests that in these seedlings considerable inactive carboxylase must be stored in a form not activatable in extracts by CO₂ and Mg²⁺. Possible mechanisms of regulation of photosynthesis by the ribulose bisphosphate carboxylase must consider not only the amount of active enzyme, but the amount of enzyme which the plant can make activatable upon demand.     

Feedback limitation of photosynthesis of Phaseolus vulgaris L grown in elevated CO2

The capacity for photosynthesis is often affected when plants are grown in air with elevated CO₂ partial pressure. We grew Phaseolus vulgaris L. in 35 and 65 Pa CO₂ and measured photosynthetic parameters. When assayed at the growth CO₂ level, photosynthesis was equal in the two CO₂ treatments. The maximum rate of ribulose-1,5-bisphosphate (RuBP) consumption was lower in plants grown at 65 Pa, but the CO₂ partial pressure at which the maximum occurred was higher in the high-CO₂-grown plants, indicating acclimation to high CO₂. The acclimation of RuBP consumption to CO₂ involved a reduction of the activity of RuBP carboxylase which resulted from reduced carbamylation, not a loss of protein. The rate of RuBP consumption declined with CO₂ when the CO₂ partial pressure was above 50Pa in plants grown under both CO₂ levels. This was caused by feedback inhibition as judged by a lack of response to removing O₂ from the air stream. The rate of photosynthesis at high CO₂ was lower in the high-CO₂-grown plants and this was correlated with reduced activity of sucrose-phosphate synthase. This is only the second report of O₂-insensitive photosynthesis under growth conditions for plants grown in high CO₂.     

Salinity and Nitrogen Effects on Photosynthesis, Ribulose-1,5-Bisphosphate Carboxylase and Metabolite Pool Sizes in Phaseolus vulgaris L

Salinity (100 millimolar NaCl) was found to reduce photosynthetic capacity independent of stomatal closure in Phaseolus vulgaris. This reduction was shown to be a consequence of a reduction in the efficiency of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) rather than a reduction in the leaf content of photosynthetic machinery. In control plants, photosynthesis became RuBP-limited at approximately 1.75 moles RuBP per mole 2-carboxyarabinitol bisphosphate binding sites. Salinization caused the RuBP pool size to reach this limiting value for CO₂ fixation at much lower values of intercellular CO₂. Plants grown at low nitrogen and ± NaCl became RuBP limited at similar RuBP pool sizes as the high nitrogen-grown plants. At limiting RuBP pool sizes and equal values of intercellular CO₂ photosynthetic capacity of salt-stressed plants was less than control plants. This effect of salinity on RuBPCase activity could not be explained by deactivation of the enzyme or inhibitor synthesis. Thus, salinity reduced photosynthetic capacity by reducing both the RuBP pool size by an effect on RuBP regeneration capacity and RuBPCase activity by an unknown mechanism when RuBP was limiting.     

Oxygen and electron flow in C4 photosynthesis: Mehler reaction, photorespiration and CO2 concentration in the bundle sheath

The photosynthetic linear electron transport rate in excess of that used for CO₂ reduction was evaluated in Sorghum bicolor Moench. [NADP-malic enzyme (ME)-type C₄ plant], Amaranthus cruentus L. (NAD-ME-type C₄ plant) and Helianthus annuus L. (C₃ plant) leaves at different CO₂ and O₂ concentrations. The electron transport rate (J _F) was calculated from fluorescence using the light partitioning factor (relative PSII cross-section) determined under conditions where excess electron transport was assumed to be negligible: low light intensities, 500 μmol CO₂ · mol⁻¹ and 2% O₂. Under high light intensities there was a large excess of J _F/4 at 10–100% O₂ in the C₃ plant due to photorespiration, but very little in sorghum and somewhat more in amaranth, showing that photorespiration is suppressed, more in the NADP-ME- and less in the NAD-ME-type species. It is concluded that when C₄ photosynthesis is limited by supply of atmospheric CO₂ to the C₄ cycle, the C₃ cycle becomes limited by regeneration of ribulose 1,5-bisphosphate (RuBP) which in turn limits RuBP oxygenase activity and photorespiration. The rate of excess electron transport over that consumed for CO₂ fixation in C₄ plants was very sensitive to the presence of O₂ in the gas phase, rapidly increasing between 0.01 and 0.1% O₂, and at 2% O₂ it was about two-thirds of that at 21% O₂. This shows the importance of the Mehler O₂ reduction as an electron sink, compared with photorespiration in C₄ plants. However, the rate of the Mehler reaction is still too low to fully account for the extra ATP which is needed in C₄ photosynthesis. Received: 8 November 1997 / Accepted: 26 December 1997     

Growth at elevated CO2: photosynthetic responses mediated through Rubisco

Abstract. The global uptake of CO₂ in photosynthesis is about 120 gigatons (Gt) of carbon per year. Virtually all passes through one enzyme, ribulose bisphosphate carboxylase/oxygenase (rubisco), which initiates both the photosynthetic carbon reduction, and photorespiratory carbon oxidation, cycles. Both CO₂ and O₂ are substrates; CO₂ also activates the enzyme. In C₃ plants, rubisco has a low catalytic activity, operates below its K_m (CO₂), and is inhibited by O₂. Consequently, increases in the CO₂/O₂ ratio stimulate C₃ photosynthesis and inhibit photorespiration. CO₂ enrichment usually enhances the productivity of C₃ plants, but the effect is marginal in C₄ species. It also causes acclimation in various ways: anatomically, morphologically, physiologically or biochemically. So, CO₂ exerts secondary effects in growth regulation, probably at the molecular level, that are not predictable from its primary biochemical role in carboxylation. After an initial increase with CO₂ enrichment, net photosynthesis often declines. This is a common acclimation phenomenon, less so in field studies, that is ultimately mediated by a decline in rubisco activity, though the RuBP/P_i-regeneration capacities of the plant may play a role. The decline is due to decreased rubisco protein, activation state, and/or specific activity, and it maintains the rubisco fixation and RuBP/P_i regeneration capacities in balance. Carbohydrate accumulation is sometimes associated with reduced net photosynthesis, possibly causing feedback inhibition of the RuBP/P_iregeneration capacities, or chloroplast disruption. As exemplified by field-grown soybeans and salt marsh species, a reduction in net photosynthesis and rubisco activity is not inevitable under CO₂ enrichment. Strong sinks or rapid translocation may avoid such acclimation responses. Over geological time, aquatic autotrophs and terrestrial C₄ and CAM plants have genetically adapted to a decline in the external CO₂/O₂ ratio, by the development of mechanisms to concentrate CO₂ internally; thus circumventing O₂ inhibition of rubisco. Here rubisco affinity for CO₂ is less, but its catalytic activity is greater, a situation compatible with a high-CO₂ internal environment. In aquatic autotrophs, the CO₂ concentrating mechanisms acclimate to the external CO₂, being suppressed at high-CO₂. It is unclear, whether a doubling in atmospheric CO₂ will be sufficient to cause a de-adaptive trend in the rubisco kinetics of future C₃ plants, producing higher catalytic activities.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium,Chromatium vinosum: I. Inducible formation

The nature of the inducible formation of enzymes engaged in the photosynthetic CO₂ fixation was examined in Chromatium vinosum during its autotropic development. Although the activity of RuBP carboxylase was the lowest among several enzyme activities examined, it was enhanced 2.5 times during a 5-hr incubation, while other enzyme activities were little altered. The enhancement of the RuBP carboxylase activity was dependent on the presence of reduced sulfur compounds in the incubation medium and illumination (>100 lx). The increase in enzyme activity, however, was repressed by CO₂ or pyruvate. Furthermore, O₂ markedly reduced the enzyme activity. In order to prove whether or not the enhancement of RuBP carboxylase activity was attributable to the biosynthesis of the enzyme, the incorporation of [³⁵S]methionine into RuBP carboxylase was followed by immunoprecipitation analysis. The incorporation was dependent on the reduced sulfur compounds, and was repressed by elevating the CO₂ level.     

Measurement of the Enzyme-CO(2)-Mg Form of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

When the amount of activation of ribulose 1,5-bisphosphate carboxylase has been measured, two forms of the enzyme, not one, are actually determined experimentally. Only the enzyme-activator CO₂-Mg²⁺ form can bind ribulose bisphosphate for reaction with substrate CO₂ or O₂. A method is presented which measures only this catalytically active form by stabilizing it with ribulose bisphosphate just before dilution and assay in Mg²⁺-free reaction medium.     

Photosynthetic CO(2) Fixation at Air Levels of CO(2) by Isolated Spinach Chloroplasts

A system has been developed for the study of photosynthetic CO₂ fixation by isolated spinach chloroplasts at air levels of CO₂. Rates of CO₂ fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO₂ fixation, suggesting that the decline in CO₂ fixation may have been caused by deactivation of the enzyme.     

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo: Control by High CO2 Concentration

High CO₂ concentrations (HC) in air induce partial deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39). Under saturating irradiance, increase in [CO₂] to 1 200 cm³ m^–3 reduces the concentration of operating carboxylation centres by 20–30 %. At a further increase in [CO₂], the activity remained on the same level. Under limiting irradiance, the lowest activity was reached at 600 cm³(CO₂) m^–3. The presence of oxygen diminished deactivation, but O₂ failed to stimulate reactivation under high CO₂. Conditions that favour oxygenation of ribulose-1,5-bisphosphate (RuBP) facilitated reactivation. Even HC did not act as an inhibitor. HC induces deactivation of RuBPCO by increasing the concentration of free reaction centres devoid of the substrate, which are more vulnerable to inhibition than the centres filled with substrates or products.     

Benzylaminopurine induced changes in photosynthesis and photorespiration of barley plants

Benzylaminopurine (BA) caused an enhancement of chlorophyll and protein content and a reduced elongation of primary barley leaves. BA did not change the rhythmic pattern of¹⁴CO₂ fixation and activities of RuBP carboxylase, RuBP oxygenase, glycolate oxidase and phosphoglycolate phosphatase, but the enzyme activities were enhanced and the level of¹⁴CO₂ fixation was reduced. Light/dark¹⁴CO₂ evolution ratio was affeoted by BA only in older leaves. BA acts sequentially on the activities of photosynthetic and photorespiratory enzymes.     

The effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1