Exchange of (18)O in the reaction of peroxynitrite with CO(2)

We have observed an exchange of (18)O in the reactions of CO(2) with peroxynitrite using membrane-inlet mass spectrometry and HPLC negative electrospray ionization mass spectrometry. The exchange appeared on addition of peroxynitrite to a solution containing (18)O-labeled CO(2) in equilibrium with bicarbonate. It was observed as a temporarily enhanced rate of depletion of (18)O from CO(2), a rate that was greater than the rate of (18)O depletion caused by the hydration/dehydration cycle of CO(2). In addition, we detected the appearance of mass peaks attributed to (18)O in product NO(3)(-).As a further measure of the (18)O exchange, there was a redistribution of (18)O such that the ratio of doubly to singly labeled CO(2) could not be described by the binomial expansion. This is not due to the hydration/dehydration cycle of CO(2) but most likely to recycling of CO(2) in the reaction with peroxynitrite. This (18)O exchange associated with the reactions of CO(2) and peroxynitrite may open a new methodology for studying this significant process.     

Oxygen and carbon dioxide mass transfer and the aerobic, autotrophic cultivation of moderate and extreme thermophiles: a case study related to the microbial desulfurization of coal

Mass transfers of O(2), CO(2), and water vapor are among the key processes in the aerobic, autotrophic cultivation of moderate and extreme thermophiles. The dynamics and kinetics of these processes are, in addition to the obvious microbial kinetics, of crucial importance for the industrial desulfurization of high-pyritic coal by such thermophiles. To evaluate the role of the temperature on the gas mass transfer, k(L)a measurements have been used to supplement the existing published data. Oxygen mass transfer from gas (air) to liquid (5 mM H(2)SO(4) in water) phase as a function of the temperature has been studied in a laboratory-scale fermentor. At 15, 30, 45, and 70 degrees C, (k(L)a)(o) values (for oxygen) were determined under three different energy input conditions by the dynamic gassing in/out method. The (k(L)a)(o) was shown to increase under these conditions with increasing temperature, and straight lines were obtained when the logarithm of (k(L)a)(o) was plotted against the temperature. By multiplying the equilibrium concentration of O(2) in water with (k(L)a)(o) maximal, O(2) transfer capacities were calculated. It appeared that in finite of a decreased solubility of O(2) at elevated temperature in mechanically mixed fermentors the calculated transfer capacities showed only minor changes for the range between 15 and 70 degrees C. However, in an air-mixed fermentor the transfer capacity of O(2) decreased slowly but steadily.Carbon dioxide mass transfer was predicted by calculations on the basis of the data for oxygen transfer. The maximal CO(2) transfer capacity, calculated as the product of the equilibrium CO(2) concentration times (k(L)a)(c), decreased slowly as the temperature increased over the range 15-70 degrees C under all three energy input conditions. Subsequent process design calculations showed that for aerobic, autotrophic cultures, CO(2) limitation is more likely to occur than O(2) limitation.     

Development of a strategy to control the dissolved concentrations of oxygen and carbon dioxide at constant shear in a plant cell bioreactor

To examine the effects of volatile components on plant cell growth, a bioreactor control system was developed to simultaneously control the dissolved concentrations of both oxygen and carbon dioxide. The first step in this work was to develop a mathematical model to account for gas-liquid mass transfer; biological utilization and production of O(2) and CO(2); and the series of chemical reactions of CO(2) in water. Using this model and dynamic measurements for dissolved O(2) and CO(2), it was observed that (1) both absorption and desorption of a volatile component could be described by a single mass transfer coefficient, K(l)a, and (2) K(l)a values for oxygen and carbon dioxide transfer were directly proportional. The second step of this work was to employ the mathematical model in an adaptive feed-forward strategy to control the dissolved O(2) and CO(2) concentrations by manipulating the inlet gas composition to the bioreactor. This strategy allowed dissolved concentrations to be controlled without the need for changing either the total gas flow rate or agitator speed. Adaptive control was required because the volumetric rates of O(2) and CO(2) consumption and production vary with time during long term operation and therefore these rates must be continually updated. As the final step, we demonstrated that this control strategy was capable of controlling the dissolved gas concentrations in both short- and long-term studies involving the cultivation of Catharanthus roseus plant cells.     

The pH dependence of the hydration of CO2 catalyzed by carbonic anhydrase III from skeletal muscle of the cat. Steady state and equilibrium studies

We have measured the pH dependence of the kinetics of CO2 hydration catalyzed by carbonic anhydrase III from the skeletal muscle of the cat. Two methods were used: an initial velocity study in which the change in absorbance of a pH indicator was measured in a stopped flow spectrophotometer, and an equilibrium study in which the rate of exchange of 18O between CO2 and H2O was measured with a mass spectrometer. We have found that the steady state constants kCO2 cat and KCO2 m are independent of pH within experimental error in the range of pH 5.0 to 8.5; the rate of release from the enzyme of the oxygen abstracted from substrate HCO-3 in the dehydration is also independent of pH in this range. This behavior is very different from that observed for carbonic anhydrase II for which kCO2 cat and the rate of release of substrate oxygen are very pH-dependent. The rate of interconversion of CO2 and HCO-3 at equilibrium catalyzed by carbonic anhydrase III is not altered when the solvent is changed from H2O to 98% D2O and 2% H2O. Thus, the interconversion probably proceeds without proton transfer in its rate-limiting steps, similar to isozymes I and II.     

Proton transfer within the active-site cavity of carbonic anhydrase III

The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.     

Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O

We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.     

Lag phase of CO2-dependent O2 evolution by illuminated Anabaena variabilis cells.

The steady-state rate of CO2-dependent O2 evolution by Anabaena variabilis cells in response to illumination was established after a lag phase. The lag phase was shortened (1) in cells incubated with glucose as an oxidizable substrate and (2) upon an increase in light intensity. The lag phase was absent during electron transfer from H2O to p-benzoquinone (in combination with ferricyanide) involving Photosystem II. A lag was observed during electron transfer from H2O to methyl viologen involving Photosystems II and I, but not for electron transfer from N,N,N',N'-tetramethyl-p-phenylenediamine (in combination with ascorbate) to methyl viologen involving only Photosystem I. The lag phases of the light-induced H2O --> CO2 and H2O --> methyl viologen electron transfer reactions showed the same temperature dependences at 10-30 degrees C. The lag was prevented by 3-(3,4-dichlorophenyl)-1,1-dimethylurea at concentrations that caused partial inhibition of photosynthetic O2 evolution. Retardation of cell respiration by a combination of CN- and benzylhydroxamate shortened the lag phase of the H2O --> methyl viologen electron transfer. It is concluded that the lag phase is associated with the electron transfer step between Photosystem II and Photosystem I common for the photosynthetic and respiratory chains and is due to the stimulation of cell respiration during the initial period of illumination as a consequence of an increase in the reduced plastoquinone pool and to subsequent retardation of respiration resulting from the transition of the electron transfer chain to the competitive pathway involving Photosystem I.     

Inorganic Carbon-Stimulated O2 Photoreduction Is Suppressed by NO2- Assimilation in Air-Grown Cells of Synechococcus UTEX 625

The effect of NO2- assimilation on O2 exchange and CO2 fixation of the cyanobacterium, Synechococcus UTEX 625, was studied mass spectrometrically. Upon addition of 1 mM inorganic carbon to the medium, inorganic carbon pools developed and accelerated O2 photoreduction 5-fold when CO2 fixation was inhibited. During steady-state photosynthesis at saturating light, O2 uptake represented 32% of O2 evolution and balanced that portion of O2 evolution that could not be accounted for by CO2 fixation. Under these conditions, NO2- assimilation reduced O2 uptake by 59% but had no influence on CO2 fixation. NO2- assimilation decreased both CO2 fixation and O2 photoreduction at low light and and increased net O2 evolution at all light intensities. The increase in net O2 evolution observed during simultaneous assimilation of carbon and nitrogen over carbon alone was due to a suppression of O2 photoreduction by NO2- assimilation. When CO2 fixation was precluded, NO2- assimilation inhibited O2 photoreduction and stimulated O2 evolution. When the electron supply was limiting (low light), competition among O2, CO2, and NO2- for electrons could be observed, but when the electron supply was not limiting (saturating light), O2 photoreduction and/or NO2- reduction caused electron transport that was additive to that for maximum CO2 fixation.     

Evidence against bicarbonate bound in the O2-evolving complex of photosystem II

The oxidation of water to molecular oxygen by photosystem II (PSII) is inhibited in bicarbonate-depleted media. One contribution to the inhibition is the binding of bicarbonate to the non-heme iron, which is required for efficient electron transfer on the electron-acceptor side of PSII. There are also proposals that bicarbonate is required for formation of O 2 by the manganese-containing O 2-evolving complex (OEC). Previous work indicates that a bicarbonate ion does not bind reversibly close to the OEC, but it remains possible that bicarbonate is bound sufficiently tightly to the OEC that it cannot readily exchange with bicarbonate in solution. In this study, we have used NH 2OH to destroy the OEC, which would release any tightly bound bicarbonate ions from the active site, and mass spectrometry to detect any released bicarbonate as CO 2. The amount of CO 2 per PSII released by the NH 2OH treatment is observed to be comparable to the background level, although N 2O, a product of the reaction of NH 2OH with the OEC, is detected in good yield. These results strongly argue against tightly bound bicarbonate ions in the OEC.     

Oxidation of deuteroferrihaem by hydrogen peroxide

1. The oxidation of deuteroferrihaem by H(2)O(2) to bile pigment and CO was studied both by stopped-flow kinetic spectrophotometry and mass spectrometry, at 25 degrees C, I=0.1m. 2. Spectrophotometric studies imply that, at constant pH, the rate of bile pigment formation is first-order with respect to [H(2)O(2)] and also proportional to [deuteroferrihaem monomer]. The effect of pH on the apparent second-order rate constant suggests that acid-ionization of deuteroferrihaem monomer is important in the reaction mechanism. 3. The relative rates of formation of O(2) (from catalytic decomposition of H(2)O(2)) and CO (from oxidation of ferrihaem) have been measured by mass spectrometry. The results are in excellent agreement with those obtained by combining kinetic data for catalytic decomposition (Jones et al., 1973, preceding paper) with the spectrophotometric results for deuteroferrihaem oxidation.     

Partitioning of the Leaf CO2 Exchange into Components Using CO2 Exchange and Fluorescence Measurements

Photorespiration was calculated from chlorophyll fluorescence and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetics and compared with CO2 evolution rate in the light, measured by three gas-exchange methods in mature sunflower (Helianthus annuus L.) leaves. The gas-exchange methods were (a) postillumination CO2 burst at unchanged CO2 concentration, (b) postillumination CO2 burst with simultaneous transfer into CO2-free air, and (c) extrapolation of the CO2 uptake to zero CO2 concentration at Rubisco active sites. The steady-state CO2 compensation point was proportional to O2 concentration, revealing the Rubisco specificity coefficient (Ksp) of 86. Electron transport rate (ETR) was calculated from fluorescence, and photorespiration rate was calculated from ETR using CO2 and O2 concentrations, Ksp, and diffusion resistances. The values of the best-fit mesophyll diffusion resistance for CO2 ranged between 0.3 and 0.8 s cm-1. Comparison of the gas-exchange and fluorescence data showed that only ribulose-1,5-bisphosphate (RuBP) carboxylation and photorespiratory CO2 evolution were present at limiting CO2 concentrations. Carboxylation of a substrate other than RuBP, in addition to RuBP carboxylation, was detected at high CO2 concentrations. A simultaneous decarboxylation process not related to RuBP oxygenation was also detected at high CO2 concentrations in the light. We propose that these processes reflect carboxylation of phosphoenolpyruvate, formed from phosphoglyceric acid and the subsequent decarboxylation of malate.     

Sensing and transfer of respiratory gases at the fish gill

The gill is both a site of gas transfer and an important location of chemoreception or gas sensing in fish. While often considered separately, these two processes are clearly intricately related because the gases that are transferred between the ventilatory water and blood at the gill are simultaneously sensed by chemoreceptors on, and within, the gill. Modulation of chemoreceptor discharge in response to changes in O(2) and CO(2) levels, in turn, is believed to initiate a series of coordinated cardiorespiratory reflexes aimed at optimising branchial gas transfer. The past decade has yielded numerous advances in terms of our understanding of gas transfer and gas sensing at the fish gill, particularly concerning the transfer and sensing of carbon dioxide. In addition, recent research has moved from striving to construct a single model that covers all fish species, to recognition of the considerable inter-specific variation that exists with respect to the mechanics of gas transfer and the cardiorespiratory responses of fish to changes in O(2) and CO(2) levels. The following review attempts to integrate gas transfer and gas sensing at the fish gill by exploring recent advances in these areas.     

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (I. Time Course of Induction of the Two Systems)

Changes in the physiological properties of the green alga Chlorella ellipsoidea (UTEX 20) were determined during adaptation from high CO2 to air. Cells of C. ellipsoidea, grown in high CO2, had an extremely low affinity for dissolved inorganic carbon (DIC). However, high-affinity DIC transport was induced rapidly after switching to air, which caused a massive decrease in the DIC concentration in the medium. Rates of O2 evolution without added carbonic anhydrase (CA) were compared with calculated rates of uncatalyzed CO2 formation in the medium as a measure of active HCO3-uptake. Cells were found to be able to use HCO3- after 5 h of adaptation and this capacity increased during the next 17 h. The stimulation of O2 evolution upon CA addition was used as a measurement of active CO2 transport: such stimulation occurred 2 h after transfer and increased during the next 5 h. Increases in O2 evolution rates were correlated closely with an increasing capacity to accumulate intracellular pools of acid-labile DIC and with decreases in K1/2(CO2) and CO2-compensation point of the cells. Treatment of cells with cycloheximide (5 [mu]g mL-1) during adaptation completely inhibited DIC transport induction, whereas treatment with chloramphenicol (400 [mu]g mL-1) had no effect, indicating the requirement for cytoplasmic protein synthesis in the induction. These results suggest that both CO2 and HCO3- transport are induced upon transfer of cells from high CO2 to air and that there is a temporal separation between the induction of the two systems.     

Kinetics of CO2 hydration in fermentors: pH and pressure effects

Fluctuations in pH and head-space pressure in a fermentor introduce temporary changes in off-gas CO(2) concentrations. These changes are quantified using a simple model based on kinetics of CO(2) hydration and gas-liquid mass transfer. The model is verified experimentally. An eigenvalue analysis of the model indicates that mass transfer is the parameter which controls the dynamics of CO(2) equilibration.     

生物氧化 CO2释放与氧化磷酸化生成 ATP 的相关性

通过解析葡萄糖有氧氧化过程中有机酸脱羧生成CO2的全部O原子来源,以及呼吸链一氧化磷酸化生成ATP的葡萄糖以外H原子的来源,明晰了葡萄糖有氧氧化过程中直接或者间接加H2O的特殊意义：H2O的H原子进入呼吸链一氧化磷酸化释放能量生成ATP;O原子结合到中间产物的C原子上形成羧基一COOH,以有机酸脱酸形式生成CO2释放出来。     

Effects of Carbon dioxide on the Rheological behavior and oxygen transfer in submerged penicillin fermentations

Fermentations of Penicillium chrysogenum have been made with different CO(2) contents in the influent gas streams. The rheological behavior of the culture broth was found to be significantly changed by exposure to high levels of CO(2). This is attributed to the wide variation in the morphology of P. chrysogenum, from normal mycelia with long hyphae to roughly spherical pellets when subjected to high levels of CO(2). A correlation has been developed relating volumetric O(2) transfer coefficients, k(L)a, with the effective O(2) diffusion coefficients, D(e), and the apparent viscosities, mu(app), based on the results obtained in this study. The use of CO(2) as a potent means for altering the rheological properties of culture broths and consequently improving the O(2) transfer capabilities in penicillin fermentations was clearly demonstrated.     

Interactions of hydrogen peroxide with ribulose bisphosphate carboxylase oxygenase

Hydrogen peroxide inhibited both carboxylase and oxygenase activities of purified, and fully activated, spinach ribulose-1,5-bisphosphate (RuP2) carboxylase-oxygenase. Inhibition of the carboxylase reaction was mixed competitive with respect to CO2 (Ki = 1.2 mM) and uncompetitive with respect to RuP2. For the oxygenase reaction, H2O2 was a competitive inhibitor with respect to O2 (Ki = 2.1 mM) and an uncompetitive inhibitor with respect to RuP2. H2O2 did not alter the stoichiometry between CO2 and RuP2 in the carboxylase reaction, indicating that H2O2 was not itself a substrate for the enzyme. RuP2 decreased the rate of deactivation of the enzyme which occurred at limiting CO2 concentrations. H2O2 greatly enhanced this stabilizing effect of RuP2 but had no effect on the rate of deactivation in the absence of RuP2. The inhibitory and stabilizing effects of H2O2 varied similarly with H2O2 concentration. These instantaneous, reversible effects of H2O2 were readily distinguishable from an irreversible inhibitory effect which occurred quite slowly, and in the absence of RuP2. These observations are discussed in relation to the enzyme's catalytic mechanism and its activation-deactivation transformations.     

Metabolic response of Platynota stultana pupae during and after extended exposure to elevated CO(2) and reduced O(2) atmospheres

The metabolic response of Platynota stultana pupae to elevated CO(2) and reduced O(2) atmospheres was measured using microcalorimetry. Initial measurements at 20 degrees C immediately upon placement in controlled atmosphere indicated a decrease in metabolic heat rate (MHR) of 27, 45, 56, 56, and 72% in an atmosphere of 5, 10, 20, 40, and 79% CO(2), respectively, and a decrease of 20, 50, 66 and 100% under 6, 2, 1, and 0% O(2). With extended exposure to controlled atmospheres, MHR increased under 5, 10, and 20% CO(2) and 6 and 2% O(2); however, the increase was greater and occurred more rapidly with lower CO(2) and higher O(2) concentration. The MHR at 40 and 79% CO(2) remained at the initial reduced level for 8 and 6 days, respectively, then decreased with longer exposure. The MHR of pupae held under 1 and 0% O(2) remained at the initial reduced level for 22 days. Upon transfer to air, the MHR of pupae increased from the reduced levels and then decreased. When the MHR decreased by no more than 30%, as a result of controlled atmosphere treatment, the pupae still developed into adults. However, when the MHR decreased by more than 50%, the energy supply was insufficient and the pupae died. Pupa mortality was comparable between 5% CO(2) and 6% O(2), and 10% CO(2) and 2% O(2). The MHR was reduced less under 20% CO(2) than under 2 or 1% O(2); however, the pupae were more susceptible to 20% CO(2) than 2 or 1% O(2). These and other data indicate an increased toxicity of high CO(2) over low O(2) atmospheres that may be related to an increase in membrane permeability as a result of CO(2) treatment.     

Molecular basis of the oxygen exchange from CO2 catalyzed by carbonic anhydrase III from bovine skeletal muscle

The exchange of 18O from CO2 to H2O in aqueous solution is caused by the hydration-dehydration cycle and is catalyzed by the carbonic anhydrases. In our previous studies of 18O exchange at chemical equilibrium catalyzed by isozymes I and II of carbonic anhydrase, we observed simple first-order depletion of 18O from CO2 with the 18O distribution among the species C18O18O, C16O18O, and C16O16O described by the binomial expansion (i.e., a random distribution of 18O). Using membrane-inlet mass spectrometry, we have measured 18O exchange between CO2 and H2O catalyzed by native zinc-containing and cobalt(II)-substituted carbonic anhydrase III from bovine skeletal muscle near pH 7.5. The distributions of 18O in CO2 deviate from the binomial expansion and are accompanied by biphasic 18O-exchange patterns; moreover, we observed regions in which 18O loss from CO2 was faster than 18O loss from HCO3-. These data are interpreted in terms of a model that includes 18O loss from an enzyme-substrate or intermediate complex. We conclude that more than one 18O can be lost from CO2 per encounter with the active site of isozyme III, a process that requires scrambling of oxygens in a bicarbonate-enzyme complex and cycling between intermediate complexes. This suggests that the rate of dissociation of H2(18)O (or 18OH-) from isozyme III is comparable to or faster than substrate and product dissociation.     

An efficient system for carbonation of high-rate algae pond water to enhance CO2 mass transfer

High-rate algal ponds have the potential to produce 59 T of dry biomass ha(-1)year(-1) based on the specific productivity of 20 g m(-2) day(-1). Atmospheric air provides only 5% of the CO(2) to the pond surface required for photosynthesis. Hence, CO(2) is usually provided via bubbling of concentrated CO(2)-air mixture into the algae ponds. This process is, however, not significantly effective in terms of mass transfer. Use of bubble column to increase the interfacial area of contact available for gas exchange is proposed as an efficient alternative. A carbonation column (CC) was modeled and designed to measure CO(2) absorptivity in-pond water at various pH regimes. The CC performed at 83% CO(2) transfer efficiency. An air-to-pond mass transport coefficient of 0.0037 m min(-1) was derived. The proposed device can be used with any exhaust gas stream with higher concentrations of CO(2) in conjunction with raceways for optimizing algae production.