Can isotopic fractionation during respiration explain the 13C-enriched sporocarps of ectomycorrhizal and saprotrophic fungi?

The mechanism behind the (13)C enrichment of fungi relative to plant materials is unclear and constrains the use of stable isotopes in studies of the carbon cycle in soils. Here, we examined whether isotopic fractionation during respiration contributes to this pattern by comparing delta(13)C signatures of respired CO(2), sporocarps and their associated plant materials, from 16 species of ectomycorrhizal or saprotrophic fungi collected in a Norway spruce forest. The isotopic composition of respired CO(2) and sporocarps was positively correlated. The differences in delta(13)C between CO(2) and sporocarps were generally small, < +/-1 per thousand in nine out of 16 species, and the average shift for all investigated species was 0.04 per thousand. However, when fungal groups were analysed separately, three out of six species of ectomycorrhizal basidiomycetes respired (13)C-enriched CO(2) (up to 1.6 per thousand), whereas three out of five species of polypores respired (13)C-depleted CO(2) (up to 1.7 per thousand; P < 0.05). The CO(2) and sporocarps were always (13)C-enriched compared with wood, litter or roots. Loss of (13)C-depleted CO(2) may have enriched some species in (13)C. However, that the CO(2) was consistently (13)C-enriched compared with plant materials implies that other processes must be found to explain the consistent (13)C-enrichment of fungal biomass compared with plant materials.     

13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase

Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe 13C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO2 in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the 13CO NMR resonance; however, the chemical shift of the 13CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The 13CO line broadening occurs with a rate constant (1080 s-1 at 20 degrees C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between 13CO and CODH-bound 13CO2 because 13CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the 13CO2 line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between 12CO and 13CO2 in solution was shown to occur at the same rate as that of CO2 reduction, which is approximately 750-fold slower than the rate of 13CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN-) was also probed by 13C NMR. A functional C-cluster is not required for 13CN- broadening (unlike for 13CO), and its exchange rate constant is 30-fold faster than that for 13CO. The combined results indicate that the 13CO exchange includes migration of CO to the C-cluster, and CO oxidation to CO2, but not release of CO2 or protons into the solvent. They also provide strong evidence of a CO2 binding site and of an internal proton transfer network in CODH. 13CN- exchange appears to monitor only movement of CN- between solution and its binding to and release from CODH.     

Influence of carbon dioxide on Nosema apis infection of honeybees (Apis mellifera)

Young workers of the honeybee Apis mellifera carnica were individually inoculated with Nosema apis spores subjected to carbon dioxide (CO(2)) treatment. The spores were kept in a CO(2) atmosphere for 30, 35 and 40 h. The course of the infection was evaluated on the basis of the survival rate of bee workers and the number of N. apis spores in their digestive tracts. CO(2) treatment of N. apis spores resulted in faster proliferation of the parasite as well as higher mortality among workers infected with spores kept in CO(2) for 30 and 35 h.     

Temporal dynamics of the carbon isotope composition in a Pinus sylvestris stand: from newly assimilated organic carbon to respired carbon dioxide

The (13)C isotopic signature (C stable isotope ratio; delta(13)C) of CO(2) respired from forest ecosystems and their particular compartments are known to be influenced by temporal changes in environmental conditions affecting C isotope fractionation during photosynthesis. Whereas most studies have assessed temporal variation in delta(13)C of ecosystem-respired CO(2) on a day-to-day scale, not much information is available on its diel dynamics. We investigated environmental and physiological controls over potential temporal changes in delta(13)C of respired CO(2) by following the short-term dynamics of the (13)C signature from newly assimilated organic matter pools in the needles, via phloem-transported organic matter in twigs and trunks, to trunk-, soil- and ecosystem-respired CO(2). We found a strong 24-h periodicity in delta(13)C of organic matter in leaf and twig phloem sap, which was strongly dampened as carbohydrates were transported down the trunk. Periodicity reappeared in the delta(13)C of trunk-respired CO(2), which seemed to originate from apparent respiratory fractionation rather than from changes in delta(13)C of the organic substrate. The diel patterns of delta(13)C in soil-respired CO(2) are partly explained by soil temperature and moisture and are probably due to changes in the relative contribution of heterotrophic and autotrophic CO(2) fluxes to total soil efflux in response to environmental conditions. Our study shows that direct relations between delta(13)C of recent assimilates and respired CO(2) may not be present on a diel time scale, and other factors lead to short-term variations in delta(13)C of ecosystem-emitted CO(2). On the one hand, these variations complicate ecosystem CO(2) flux partitioning, but on the other hand they provide new insights into metabolic processes underlying respiratory CO(2) emission.     

Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions

For estimating the oxidation rates (Rox) of glucose and other substrates by use of (13)C-labeled tracers, we obtained correction factors to account for label dilution in endogenous bicarbonate pools and TCA cycle exchange reactions. Fractional recoveries of (13)C label in respiratory gases were determined during 225 min of rest and 90 min of leg cycle ergometry at 45 and 65% peak oxygen uptake (VO(2 peak)) after continuous infusions of [1-(13)C]acetate, [2-(13)C]acetate, or NaH(13)CO(3). In parallel trials, [6,6-(2)H]glucose and [1-(13)C]glucose were given. Experiments were conducted after an overnight fast with exercise commencing 12 h after the last meal. During the transition from rest to exercise, CO(2) production increased (P < 0.05) in an intensity-dependent manner. Significant differences were observed in the fractional recoveries of (13)C label as (13)CO(2) at rest (NaH(13)CO(3), 77.5 +/- 2.8%; [1-(13)C]acetate, 49.8 +/- 2.4%; [2-(13)C]acetate, 26.1 +/- 1.4%). During exercise, fractional recoveries of (13)C label from [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO(3) were increased compared with rest. Magnitudes of label recoveries during both exercise intensities were tracer specific (NaH(13)CO(3), 93%; [1-(13)C]acetate, 80%; [2-(13)C]acetate, 65%). Use of an acetate-derived correction factor for estimating glucose oxidation resulted in Rox values in excess (P < 0.05) of glucose rate of disappearance during hard exercise. We conclude that, after an overnight fast: 1) recovery of (13)C label as (13)CO(2) from [(13)C]acetate is decreased compared with bicarbonate; 2) the position of (13)C acetate label affects carbon dilution estimations; 3) recovery of (13)C label increases in the transition from rest to exercise in an isotope-dependent manner; and 4) application of an acetate correction factor in glucose oxidation measurements results in oxidation rates in excess of glucose disappearance during exercise at 65% of VO(2 peak). Therefore, bicarbonate, not acetate, correction factors are advocated for estimating glucose oxidation from carbon tracers in exercising men.     

Metabolic origin of carbon isotope composition of leaf dark-respired CO2 in French bean

The carbon isotope composition (delta(13)C) of CO(2) produced in darkness by intact French bean (Phaseolus vulgaris) leaves was investigated for different leaf temperatures and during dark periods of increasing length. The delta(13)C of CO(2) linearly decreased when temperature increased, from -19 per thousand at 10 degrees C to -24 per thousand at 35 degrees C. It also progressively decreased from -21 per thousand to -30 per thousand when leaves were maintained in continuous darkness for several days. Under normal conditions (temperature not exceeding 30 degrees C and normal dark period), the evolved CO(2) was enriched in (13)C compared with carbohydrates, the most (13)C-enriched metabolites. However, at the end of a long dark period (carbohydrate starvation), CO(2) was depleted in (13)C even when compared with the composition of total organic matter. In the two types of experiment, the variations of delta(13)C were linearly related to those of the respiratory quotient. This strongly suggests that the variation of delta(13)C is the direct consequence of a substrate switch that may occur to feed respiration; carbohydrate oxidation producing (13)C-enriched CO(2) and beta-oxidation of fatty acids producing (13)C-depleted CO(2) when compared with total organic matter (-27.5 per thousand). These results are consistent with the assumption that the delta(13)C of dark respired CO(2) is determined by the relative contributions of the two major decarboxylation processes that occur in darkness: pyruvate dehydrogenase activity and the Krebs cycle.     

Carbon-13 NMR studies of 13CO binding to human hemoglobin

In the ¹³C NMR spectrum of hemoglobin A carbonylated with ¹³CO, separate resonances can be distinguished at 207.04 ppm and 206.60 ppm (with respect to the ¹³C resonance of external tetramethyl-silane) for ¹³Co bound to the α and β chains of the hemoglobin tetramer. A study of the ¹³Co derivatives of the isolated α and β chains, and of the abnormal hemoglobin M_IWATE which contains α chains which are in the met [Fe(III)] form and do not bind CO, has permitted an assignment of the high field (206.60 ppm) resonance to the β chain ¹³CO and the low field one to the α chain ¹³CO. The identification of these ¹³Co resonances permits a study of the differences in the chemistry of the α and β heme units in intact hemoglobin. Some results on the differences in the redox behavior of these chains are included.     

Phenylalanine kinetics in healthy volunteers and liver cirrhotics: implications for the phenylalanine breath test

Expired 13CO2 recovery from an oral l-[1-13C]phenylalanine ([13C]Phe) dose has been used to quantify liver function. This parameter, however, does not depend solely on liver function but also on total CO2 production, Phe turnover, and initial tracer distribution. Therefore, we evaluated the impact of these factors on breath test values. Nine ethyl-toxic cirrhotic patients and nine control subjects received intravenously 2 mg/kg of [13C]Phe, and breath and blood samples were collected over 4 h. CO2 production was measured by indirect calorimetry. The exhaled 13CO2 enrichments were analyzed by isotope ratio mass spectrometry and the [13C]Phe and l-[1-13C]tyrosine enrichments by gas chromatography-mass spectrometry. The cumulative 13CO2 recovery was significantly lower in cirrhotic patients (7 vs. 12%; P < 0.01), in part due to lower total CO2 production rates. Phe turnover in cirrhotic patients was significantly lower (33 vs. 44 micro mol. kg(-1). h(-1); P < 0.05). When these extrahepatic factors were considered in the calculation of the Phe oxidation rate, the intergroup differences were even more pronounced (3 vs. 7 micro mol. kg(-1). h(-1)) than those for 13CO2 recovery data. Also, the Phe-to-Tyr conversion rate, another indicator of Phe oxidation, was significantly reduced (0.7 vs. 3.0 micro mol. kg(-1). h(-1)).     

A 13C nuclear-magnetic-resonance study of CO2-HCO3-exchange catalyzed by human carbonic anhydrase C at chemical equilibrium.

The effects of human carbonic anhydrase C on the 13C nuclear magnetic resonance spectra of equilibrium mixtures of 13CO2 and NaH13CO3 were measured at 67.89 MHz. Enzyme-catalyzed CO2-HCO-3 exchange rates were estimated from the linewidths of the resonances. The results show that: (a) the maximal exchange rates are larger than the maximal turnover rates; (b) the exchange is equally rapid with 1H2O or with 2H2O as solvents; (c) the exchange is equally rapid in the presence or in the absence of added buffers; (d) the apparent substrate binding is weaker than predicted if steady-state Km values are assumed to represent substrate dissociation constants. The main conclusion concerning the catalytic mechanism of the enzyme is that the proton-transfer processes which limit turnover rates in the steady state are not directly involved in CO2-HCO-3 exchange. In addition, the results suggest that CO2-HCO-3 interconversion takes place by a nucleophilic mechanism, such as a reversible reaction of zinc-coordinated OH- with CO2.     

Tracing carbon monoxide uptake by Clostridium ljungdahlii during ethanol fermentation using 13C-enrichment technique

Conversion of synthesis gas (CO and H₂) to ethanol can be an alternative, promising technology to produce biofuels from renewable biomass. To distinguish microbial utilization of carbon source between fructose and synthesis gas CO and to evaluate biological production of ethanol from CO, we adopted the ¹³C-enrichment of the CO substrate and hypothesized that the residual increase in δ¹³C of the cell biomass would reflect the increased contribution of ¹³C-enriched CO. Addition of synthesis gas to live culture medium for ethanol fermentation by Clostridum ljungdahlii increased the microbial growth and ethanol production. Despite the high ¹³C-enrichment in CO (99 atom % ¹³C), however, microbial δ¹³C increased relatively small compared to the microbial growth. The uptake efficiency of CO estimated using the isotope mass balance equation was also very low: 0.0014 % for the low CO and 0.0016 % for the high CO treatment. Furthermore, the fast production of ethanol in the early stage indicated that the presence of sugar in fermentation medium would limit the utilization of CO as a carbon source by C. ljungdahlii.     

Assessment of age-related glucose oxidation rates of broiler chickens by using stable isotopes

During their relatively short commercial lifespan of six weeks, broiler chickens undergo very pronounced age- or body weight-related changes in metabolic rate and body composition. The present study was aimed to assess the age-related changes in glucose oxidation rate of broiler chickens by using 13C-labeled glucose. The methodology for this breath test needed to be established first. Broiler chickens aged from two to six weeks were placed in open-circuit respiration cells and received a single intubation of U-13C6-glucose, followed by breath sampling for 4 hours and mass spectrometric analysis of 13C: 12C ratio in the exhaled air. Simultaneously, CO2 concentration in the respiration cell air was continuously monitored in order to calculate the cumulative percentage dose recovery (CPDR). With respect to the methodology, an oral dose of 2 mg U-13C6-glucose per kg body weight while maintaining a CO2 in the concentration of 0.4 to 0.5% was considered to be optimal. The three-parameter Gompertz curve fitted the CPDR values very well. Pronounced age-related changes in exogenous glucose oxidation rates in rapidly growing meat-type chickens were assessed. Young broiler chickens spend only a relatively low percentage of ingested glucose for immediate oxidation. In contrast, broiler chickens approaching the age of maximal absolute growth rate oxidize a greater proportion of the recently ingested glucose relative to the non-oxidative disposal pathways. This shift in the exogenous partitioning is discussed in relation to age-dependent changes in glucose turnover, lipid oxidation and deposition and metabolic heat production.     

Diurnal variation of the delta 13C of pine needle respired CO2 evolved in darkness

The delta 13C of pine needle CO2 evolved in darkness (delta 13Cr) for slash pine trees (Pinus elliottii) was determined by placing recently collected pine needles in darkness and collecting respired CO2 over a short time period (<15 min). Delta 13Cr measurements were made over several 24 h periods to test the hypothesis that significant variation in delta 13Cr would be observed during a diurnal cycle. The delta 13Cr measurements from the 24 h time series trials showed a consistent midday 13C-enrichment (5-10 per thousand) relative to bulk biomass. The delta 13Cr values became more 13C-depleted at night and following shading, and approached bulk-biomass delta 13C values by dawn. The effect of night-time respired 13C-enriched CO2 on the delta 13C value of the remaining assimilate is shown to be minimal (13C depleted by 0.22 per thousand) under field conditions for P. elliottii needles.     

Mitochondrial-driven bicarbonate transport supports photosynthesis in a marine microalga

The CO(2)-concentrating mechanism (CCM) of the marine eustigmatophycean microalga Nannochloropsis gaditana consists of an active HCO(3)(-) transport system and an internal carbonic anhydrase to facilitate accumulation and conversion of HCO(3)(-) to CO(2) for photosynthetic fixation. Aqueous inlet mass spectrometry revealed that a portion of the CO(2) generated within the cells leaked to the medium, resulting in a significant rise in the extracellular CO(2) concentration to a level above its chemical equilibrium that was diagnostic for active HCO(3)(-) transport. The transient rise in extracellular CO(2) occurred in the light and the dark and was resolved from concurrent respiratory CO(2) efflux using H(13)CO(3)(-) stable isotope techniques. H(13)CO(3)(-) pump-(13)CO(2) leak activity of the CCM was unaffected by 10 microM 3(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of chloroplast linear electron transport, although photosynthetic O(2) evolution was reduced by 90%. However, low concentrations of cyanide, azide, and rotenone along with anoxia significantly reduced or abolished (13)CO(2) efflux in the dark and light. These results indicate that H(13)CO(3)(-) transport was supported by mitochondrial energy production in contrast to other algae and cyanobacteria in which it is supported by photosynthetic electron transport. This is the first report of a direct role for mitochondria in the energization and functioning of the CCM in a photosynthetic organism.     

Structure of a galactosaminoglycan from Cordyceps ophioglossoides

A water- and alkali-insoluble galactosaminoglycan (CON), precipitated with ammonium hydroxide from the culture filtrate of Cordyceps ophioglossoides, is composed mainly of 2-amino-2-deoxy-d-galactose (80.5%) together with small proportions of glucose, galactose, and mannose, protein (3.6%), and acetyl groups (1%). CON was eluted as a single peak in gel filtration, and the average molecular weight was estimated to be ～50,000. Partial, acid hydrolysis of CON gave small CON and homologous 2-amino-2-deoxy-d-galacto-oligosaccharides. Small CON (mol. wt. ～10,000) was soluble in water and composed only of 2-amino-2-deoxy-d-galactose. The results of methylation analysis, ¹³C-n.m.r. studies, and enzymic hydrolysis indicated small CON to be a (1→4)-linked 2-amino-2-deoxy-α-d-galactopyranan, and the ¹³C-n.m.r. data indicated the glycosidic linkage in the polygalactosamine moiety of CON to be the same as that of small CON.     

Metabolic-flux analysis of continuously cultured hybridoma cells using (13)CO(2) mass spectrometry in combination with (13)C-lactate nuclear magnetic resonance spectroscopy and metabolite balancing

Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO(2), and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste products derived from glucose. Continuously cultured hybridoma cells are grown in medium containing either 1-(13)C-, 2-(13)C-, or 6-(13)C-glucose. The uptake and production rates of amino acids, glucose, ammonia, O(2), and CO(2) as well as the cellular composition are measured. In addition, the (13)C distribution of the lactate produced and alanine produced by the hybridomas is determined by (1)H-NMR spectroscopy, and the (13)CO(2)/(12)CO(2) ratio is measured by on-line mass spectrometry. These data are used to calculate the intracellular fluxes of the glycolysis, the pentose phosphate pathway, the TCA cycle, and fluxes involved in amino acid metabolism. It is shown that: (i) approximately 20% of the glucose consumed is channeled through the pentose shunt; (ii) the glycolysis pathway contributes the most to lactate production, and most of the CO(2) is produced by the TCA cycle; (iii) the pyruvate-carboxylase flux is negligibly small; and (iv) the malic-enzyme flux is estimated to be 10% of the glucose uptake rate. Based on these flux data suggestions are made to engineer a more efficient glucose metabolism in mammalian cells.     

Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively 13C labeled samples

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated (13)C spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (13)CO experiments however the large separation between (13)CO and (13)C(alpha) chemical shifts offers hope that robust (13)CO dispersion profiles can be recorded on uniformly (13)C labeled samples, leading to the extraction of accurate (13)CO chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-(13)C]-pyruvate and NaH(13)CO(3,) or uniformly labeled, generated from (13)C-glucose. Very similar (13)CO chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.     

Apparent respiratory discrimination is correlated with growth rate in the shoot apex of sunflower (Helianthus annuus)

The literature offers no consensus as to whether the delta(13)C of respired CO(2) is identical to that of the respiratory substrate, perhaps because of differences in measurement technique and growth conditions. To address this issue, the delta(13)C of respired CO(2) from growing sunflower shoot apices was measured and compared with that of soluble carbohydrates extracted from the respiring tissues. Shoot apices were studied because any influence of growth and biosynthesis was expected to be maximally expressed in these rapidly growing tissues. The two most probable substrates, starch and soluble sugars, were similar in delta(13)C (P=0.46). The delta(13)C of respired CO(2) was enriched in (13)C compared with these putative substrates (P<0.0001). This apparent enrichment ranged from 2.2 per thousand-5.7 per thousand, and decreased with relative growth rate (P<0.0001). The respiratory enrichment was counterbalanced by a depletion in the tissue constructed from the residual carbohydrates. The depletion varied from 2.2 per thousand to 3.0 per thousand relative to soluble carbohydrates (P<0.05), as predicted from mass-balance arguments. These results support the idea that respired CO(2) is enriched relative to its substrates. Variation in growth rates may help to explain the variable amounts of respiratory discrimination described in the literature.     

The CO and CN(-) ligands to the active site Fe in [NiFe]-hydrogenase of Escherichia coli have different metabolic origins

The Fe atom in the bimetallic active site of [NiFe]-hydrogenases has one CO and two cyanide ligands. To determine their metabolic origin, [NiFe]-hydrogenase-2 was isolated from Escherichia coli grown in the presence of L-[ureido-(13)C]citrulline, purified and analyzed by infrared spectroscopy. The spectra indicate incorporation of (13)C only into the cyanide ligands and not into the CO, showing that cyanide and CO have different metabolic origins. After growth of E. coli in the presence of (13)CO only the CO ligand was labelled with (13)C. Labelling did not result from an exchange of the intrinsic CO ligand with the exogenous CO.     

Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle

A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.