Measuring protein synthesis using metabolic H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water (²H₂O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) [16], and we confirmed that ²H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the ²H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT–ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given ²H₂O.     

Comparison of gas chromatography–mass spectrometry and gas chromatography–combustion–isotope ratio mass spectrometry analysis for in vivo estimates of metabolic fluxes

Gas chromatography–mass spectrometry (GC–MS) was compared with gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for measurements of cholesterol ¹³C enrichment after infusion of labeled precursor ([¹³C_1,2]acetate). Paired results were significantly correlated, although GC–MS was less accurate than GC–C–IRMS for higher enrichments. Nevertheless, only GC–MS was able to provide information on isotopologue distribution, bringing new insights to lipid metabolism. Therefore, we assessed the isotopologue distribution of cholesterol in humans and dogs known to present contrasted cholesterol metabolic pathways. The labeled tracer incorporation was different in both species, highlighting the subsidiarity of GC–MS and GC–C–IRMS to analyze in vivo stable isotope studies.     

Two distinct plant respiratory physiotypes might exist which correspond to fast-growing and slow-growing species

The origin of the carbon atoms in CO₂ respired by leaves in the dark of several plant species has been studied using ¹³C/¹²C stable isotopes. This study was conducted using an open gas exchange system for isotope labeling that was coupled to an elemental analyzer and further linked to an isotope ratio mass spectrometer (EA–IRMS) or coupled to a gas chromatography–combustion-isotope ratio mass spectrometer (GC–C-IRMS). We demonstrate here that the carbon, which is recently assimilated during photosynthesis, accounts for nearly ca. 50% of the carbon in the CO₂ lost through dark respiration (R_d) after illumination in fast-growing and cultivated plants and trees and, accounts for only ca. 10% in slow-growing plants. Moreover, our study shows that fast-growing plants, which had the largest percentages of newly fixed carbon of leaf-respired CO₂, were also those with the largest shoot/root ratios, whereas slow-growing plants showed the lowest shoot/root values.     

Heavy water and 15N labelling with NanoSIMS analysis reveals growth rate‐dependent metabolic heterogeneity in chemostats

To measure single‐cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labelling of microbial populations with heavy water (a passive tracer) and ¹⁵N ammonium in combination with multi‐isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single‐cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single‐cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (～0.1 atom % ²H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (～6 h, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium versus amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.     

Site-specific isotope fractionation of hydrogen in the biosynthesis of plant fatty acids

The overall deuterium content of plant lipids has been investigated by isotope ratio mass spectrometry (IRMS), and the site-specific natural isotope fractionation of hydrogen has been studied by ²H-NMR at natural abundance (SNIF-NMR). An analytical strategy has been developed in order to exploit the isotopomeric composition determined in clusters associated with different chemical sites of one or several fatty acid components. The method, which combines spectrometric and chromatographic data, enables isotopic criteria to be directly derived from raw vegetable oils containing in general two saturated and two unsaturated fatty acids. These results provide new information on isotopic fractionation caused by biochemical, physiological and natural environmental effects. Some alternation in the molecular deuterium distribution has been detected which may be related to the mechanism of fatty acid elongation. The successive methylene groups introduced through malonyl CoA are the subjects of different kinetic isotope effects since one of them is exclusively derived from NADH whereas the other has a contribution from pyruvate. A discriminant analysis of the cluster isotopic parameters enables several kinds of botanical precursors to be distinguished. The authenticating performances can be improved by taking into account the influence of climatic effects related to the region in which the plant grew.     

Conformational changes of recombinant monoclonal antibodies by limited proteolytic digestion,stable isotope labeling,and liquid chromatography–mass spectrometry

Limited proteolytic digestion is a method with a long history that has been used to study protein domain structures and conformational changes. A method of combining limited proteolytic digestion, stable isotope labeling, and mass spectrometry was established in the current study to investigate protein conformational changes. Recombinant monoclonal antibodies with or without the conserved oligosaccharides, and with or without oxidation of the conserved methionine residues, were used to test the newly proposed method. All of the samples were digested in ammonium bicarbonate buffer prepared in normal water. The oxidized deglycosylated sample was also digested in ammonium bicarbonate buffer prepared in ¹⁸O-labeled water. The sample from the digestion in ¹⁸O–water was spiked into each sample digested in normal water. Each mixed sample was subsequently analyzed by liquid chromatography–mass spectrometry (LC–MS). The molecular weight differences between the peptides digested in normal water versus ¹⁸O–water were used to differentiate peaks from the samples. The relative peak intensities of peptides with or without the C-terminal incorporation of ¹⁸O atoms were used to determine susceptibility of different samples to trypsin and chymotrypsin. The results demonstrated that the method was capable of detecting local conformational changes of the recombinant monoclonal antibodies caused by deglycosylation and oxidation.     

Reproducibility of gas chromatography-mass spectrometry measurements of 2H labeling of water: application for measuring body composition in mice

Deuterium-labeled water (2H2O) has emerged as a novel isotope tracer. Following the administration of 2H2O, it is possible to study the dynamics of carbohydrate, protein, lipid, and DNA and to determine body composition. Those studies require reliable measurements of the 2H labeling of water. Although simple gas chromatography-mass spectrometry (GC-MS) methods have been developed for measuring the 2H enrichment of biological fluids, investigators have not reported on the intra- and/or interdaily variability of the measurements. We have experimentally examined the reproducibility of one GC-MS method for measuring the 2H labeling of water. Briefly, hydrogen (deuterium) atoms in water were exchanged with those bound to acetone, and the 2H labeling of acetone was then determined under electron impact ionization. We found that the coefficient of variation is generally less than 0.5% when water is labeled between 0 and 2.8 mole percentage excess 2H. We demonstrated that this highly reproducible result allows one to use 2H2O and the "acetone method" to measure physiological parameters such as body composition in mice.     

Isolation and derivatization of plasma taurine for stable isotope analysis by gas chromatography-mass spectrometry

A method for the isolation and derivatization of plasma taurine is described that allows stable isotope determinations of taurine to be made by gas chromatography-mass spectrometry (gc-ms). The isolation procedure can be applied to 0.1 ml of plasma: the recovery of plasma taurine was 70–80%. For gc separation, taurine was converted to its dimethylaminomethylene methyl ester derivative which could not be detected by hydrogen flame ionization, but could be monitored readily by NH₃ chemical ionization mass spectrometry. The derivatization reaction occurred partially on-column and required optimization of injection conditions. Using stable isotope ratiometry multiple ion detection, $\text{[M + 2 + H]}{}^{\mathrm{+}}\text{[M + H]}{}^{\mathrm{+}}$ ion ratio of natural abundance taurine was determined with a standard deviation of less than ±0.07% of the ratio. The [1,2-¹³C]taurine/taurine mole ratios of standard mixtures could be accurately determined to 0.001. This stable isotope gc-ms method is suitable for studying the plasma kinetics of [1,2-¹³C]taurine in infants who are at risk with respect to taurine depletion.     

The role of light in soybean seed filling metabolism

Soybean (Glycine max) yields high levels of both protein and oil, making it one of the most versatile and important crops in the world. Light has been implicated in the physiology of developing green seeds including soybeans but its roles are not quantitatively understood. We have determined the light levels reaching growing soybean embryos under field conditions and report detailed redox and energy balance analyses for them. Direct flux measurements and labeling patterns for multiple labeling experiments including [U‐¹³C₆]‐glucose, [U‐¹³C₅]‐glutamine, the combination of [U‐¹⁴C₁₂]‐sucrose + [U‐¹⁴C₆]‐glucose + [U‐¹⁴C₅]‐glutamine + [U‐¹⁴C₄]‐asparagine, or ¹⁴CO₂ labeling were performed at different light levels to give further insight into green embryo metabolism during seed filling and to develop and validate a flux map. Labeling patterns (protein amino acids, triacylglycerol fatty acids, starch, cell wall, protein glycan monomers, organic acids), uptake fluxes (glutamine, asparagine, sucrose, glucose), fluxes to biomass (protein amino acids, oil), and respiratory fluxes (CO₂, O₂) were established by a combination of gas chromatography‐mass spectrometry, ¹³C‐ and ¹H‐NMR, scintillation counting, HPLC, gas chromatography‐flame ionization detection, C:N and amino acid analyses, and infrared gas analysis, yielding over 750 measurements of metabolism. Our results show: (i) that developing soybeans receive low but significant light levels that influence growth and metabolism; (ii) a role for light in generating ATP but not net reductant during seed filling; (iii) that flux through Rubisco contributes to carbon conversion efficiency through generation of 3‐phosphoglycerate; and (iv) a larger contribution of amino acid carbon to fatty acid synthesis than in other oilseeds analyzed to date.     

Quantitative and carbon isotope ratio analysis of fatty acids isolated from human brain hemispheres

Our knowledge surrounding the overall fatty acid profile of the adult human brain has been largely limited to extrapolations from brain regions in which the distribution of fatty acids varies. This is especially problematic when modeling brain fatty acid metabolism, therefore, an updated estimate of whole-brain fatty acid concentration is necessitated. Here, we sought to conduct a comprehensive quantitative analysis of fatty acids from entire well-characterized human brain hemispheres (n = 6) provided by the Douglas-Bell Canada Brain Bank. Additionally, exploratory natural abundance carbon isotope ratio (CIR; δ¹³C, ¹³C/¹²C) analysis was performed to assess the origin of brain fatty acids. Brain fatty acid methyl esters (FAMEs) were quantified by gas chromatography (GC)-flame ionization detection and minor n-6 and n-3 polyunsaturated fatty acid pentafluorobenzyl esters by GC-mass spectrometry. Carbon isotope ratio values of identifiable FAMEs were measured by GC-combustion-isotope ratio mass spectrometry. Overall, the most abundant fatty acid in the human brain was oleic acid, followed by stearic acid (STA), palmitic acid (PAM), docosahexaenoic acid (DHA), and arachidonic acid (ARA). Interestingly, cholesterol as well as saturates including PAM and STA were most enriched in ¹³C, while PUFAs including DHA and ARA were most depleted in ¹³C. These findings suggest a contribution of endogenous synthesis utilizing dietary sugar substrates rich in ¹³C, and a combination of marine, animal, and terrestrial PUFA sources more depleted in ¹³C, respectively. These results provide novel insights on cerebral fatty acid origin and concentration, the latter serving as a valuable resource for future modeling of fatty acid metabolism in the human brain.

     

Comparison of Zinc Reduction with Platinum Reduction for Analysis of Deuterium‐Enriched Water Samples for the Doubly Labeled Water Technique

Objective: Isotope ratio mass spectrometry of hydrogen and oxygen is frequently used to determine total energy expenditure (TEE) using doubly labeled water. Conventionally, hydrogen isotope ratio is determined in hydrogen gas generated from water samples using zinc reduction. We compare this with a new automated platinum method to determine the ratios of hydrogen isotopes in deuterium‐enriched water samples. Research Methods and Procedures: The platinum method of sample preparation was compared with the zinc method in three ways: analytical variation in deuterium enrichment (within sample; n = 51), analytical variation in TEE estimates (within sample set; n = 10), and level of agreement of TEE estimates between both methods (n = 14). Results: For the zinc method, the standard deviation for multiple sets of triplicate ²H₂O sample analysis was ±4.36‰ and ±2.07‰ for platinum. The correlation between TEE estimates when sample sets were analyzed in duplicate was r = 0.89 for zinc and r = 0.83 for platinum. The intercept and slope of the regression line were significantly different from the line of identity for duplicate TEE estimates by zinc but were not different from the line of identity for platinum. After correction for the intra‐assay variation of each method, the correlation between zinc and platinum for TEE was 0.77, and the intercept, but not the slope, of the regression was significantly different from the line of identity. The mean difference between the zinc method and the platinum method was 56 kcal/day, and the 95% confidence interval was ?438 to 550 kcal/day. Discussion: These data suggest that the platinum method is at least as reliable as the zinc method as a sample preparation technique for isotope ratio mass spectrometry of deuterium‐enriched water samples. The platinum method is also less costly and less labor‐intensive than the zinc method.     

13C metabolic flux analysis for larger scale cultivation using gas chromatography-combustion-isotope ratio mass spectrometry

¹³C-based metabolic flux analysis (¹³CMFA) is limited to smaller scale experiments due to very high costs of labeled substrates. We measured ¹³C enrichment in proteinogenic amino acid hydrolyzates using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) from a series of parallel batch cultivations of Corynebacterium glutamicum utilizing mixtures of natural glucose and [1-¹³C] glucose, containing 0%, 0.5%, 1%, 2%, and 10% [1-¹³C] glucose. Decreasing the [1-¹³C] glucose content, kinetic isotope effects played an increasing role but could be corrected. From the corrected ¹³C enrichments in vivo fluxes in the central metabolism were determined by numerical optimization. The obtained flux distribution was very similar to those obtained from parallel labeling experiments using conventional high labeling GC-MS method and to published results. The GC-C-IRMS-based method involving low labeling degree of expensive tracer substrate, e.g. 1%, is well suited for larger laboratory and industrial pilot scale fermentations.     

Oxidized fatty acid analysis by charge-switch derivatization,selected reaction monitoring,and accurate mass quantitation

A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0 pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r² > 0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA₂γ^–/– mouse liver identifying the role of iPLA₂γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems.     

Variations in the natural abundance of oxygen-18 and deuterium in plant carbohydrates

Variations in the natural abundance of ¹⁸O and ²H in plant cellulose are influenced by the isotopic composition of the water directly involved in metabolism—the metabolic water fraction. The isotopic distinction between the metabolic source water and total tissue water must reflect the formation of isotopic gradients within the tissue that are influenced by the rate of water turnover, by properties of the water conducting system and by environmental conditions. It seems that the ¹⁸O abundance in the metabolic water is conserved in cellulose with a relatively constant isotope effect. The relationship of the ²H abundance between metabolic water and cellulose is more complex. Hydrogen incorporated into photosynthetic products during primary reduction steps is highly depleted in ²H. However, a large proportion of these hydrogens are subsequently replaced by exchange with water, leading to ²H enrichment during heterotrophic metabolism. Deciphering the oxygen isotope ratio of cellulose could help in providing insights into the carbon and oxygen fluxes exchanged between plants and the atmosphere. This is because the ¹⁸O abundance in cellulose records the ¹⁸O abundance in the metabolic water, which in turn, controls the oxygen isotopic signatures of the CO₂ and O₂ released by plants into the atmosphere. The hydrogen isotope effects associated with carbohydrate metabolism provide insights into the autotrophic state of a plant tissue. This is because the hydrogen isotope ratio of carbohydrates must reflect the net effects of the two opposing isotope effects associated with photosynthesis and heterotrophic metabolism.     

Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.     

Spontaneous Hydrolysis and Dehydration of Dehydroascorbic Acid in Aqueous Solution

The interaction of water with dehydroascorbic acid was examined by incubating dehydroascorbic acid and ascorbic acid in¹⁸O-labeled water for various amounts of time and then oxidizing the products with hydrogen peroxide or reducing the products with mercaptoethanol, with analysis by gas chromatography mass spectrometry. Based on mass changes, dehydroascorbic acid readily exchanged three oxygen atoms with H₂¹⁸O. When mercaptoethanol was used to reduce dehydroascorbic acid (which had been incubated in H₂¹⁸O) to ascorbic acid, the newly formed ascorbic acid also contained three labeled oxygen atoms. However, ascorbic acid incubated in H₂¹⁸O for the same amount of time under identical conditions exchanged only two labeled oxygen atoms. Electron impact mass spectrometry of derivatized ascorbic acid created a decarboxylation product which had only two labeled oxygen atoms, regardless if 3-oxygen-labeled or 2-oxygen-labeled ascorbic acid was the parent compound, isolating the extra oxygen addition to carbon 1. These data suggest that dehydroascorbic acid spontaneously hydrolyzes and dehydrates in aqueous solution and that the hydrolytic-hydroxyl oxygen is accepted by carbon 1. Ascorbic acid, on the other hand, does not show this same tendency to hydrolyze.     

Metabolic processes account for the majority of the intracellular water in log-phase Escherichia coli cells as revealed by hydrogen isotopes

It is generally believed that water transport across biological membranes is essentially a near-instantaneous process, with water molecules diffusing directly across the membrane as well as through pores such as aquaporins. As a result of these processes by which water can equilibrate across a membrane, a common assumption is that intracellular water is isotopically indistinguishable from extracellular water. To test this assumption directly, we measured the hydrogen isotope ratio of intracellular water in Escherichia coli cells. Our results demonstrate that more than 50% of the intracellular water hydrogen atoms in log-phase E. coli cells are isotopically distinct from the growth medium water and that these isotopically distinct hydrogen atoms are derived from metabolic processes. As expected, the (2)H/(1)H isotope ratio of intracellular water from log-phase cells showed an appreciably larger contribution from metabolic water than did intracellular water from stationary-phase cells (53 +/- 12 and 23 +/- 5%, respectively). The (2)H/(1)H isotope ratio of intracellular water was also monitored indirectly by measuring the isotope ratio of fatty acids, metabolites that are known to incorporate hydrogen atoms from water during biosynthesis. Significantly, the difference in the isotopic composition of intracellular water from log- to stationary-phase E. coli cells was reflected in the hydrogen isotope ratio of individual fatty acids harvested at the two different times, indicating that the isotope ratio of metabolites can be used as an indirect probe of metabolic activity. Together, these results demonstrate that contrary to the common assumption that intracellular water is isotopically identical to extracellular water, these two pools of water can actually be quite distinct.     

Regulation of malic-acid metabolism in Crassulacean-acid-metabolism plants in the dark and light: In-vivo evidence from 13C-labeling patterns after 13CO2 fixation

The labeling patterns in malic acid from dark ¹³CO₂ fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and ¹³C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12–14 h dark ¹³CO₂ fixation the ratio of [4-¹³C] to [1-¹³C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The ¹³C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87–89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following ¹³CO₂ fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [¹³C] malic acid (¹³CO₂ or [1-¹³C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to ¹³CO₂ in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284–291). In the shortest times, only singly-labeled [4-¹³C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.Abbreviations CAM Crassulacean acid metabolism - GCMS gas chromatography-mass spectrometry - MS mass spectrometry - NMR nuclear magnetic resonance spectrometry - PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate     

15N-labeled P22 c2 repressor for nuclear magnetic resonance studies of protein-DNA interactions

The salmonella phage P22 c2 repressor was produced with 90% ¹⁵N isotope labeling of all leucines, using the expression system E. coli W3110 lac I ^Q/pTP 125. The N-terminal DNA-binding domain 1–76 was obtained by chymotrypsin cleavage. Its characterization by biochemical techniques, mass spectrometry, and one- and two-dimensional nuclear magnetic resonance (NMR) showed that highly residue-selective isotope labeling was achieved with the minimal growth medium used. The ability to obtain such isotope labeling opens new avenues for NMR studies of protein-DNA interactions in the P22 operator system.     

Origin of hydrogen in methane produced by Methanobacterium thermoautotrophicum.

The production of deuterated methane by Methanobacterium thermoautotrophicum in H2O-D2O mixtures was examined by high-resolution mass spectrometry. The hydrogen in the methane arose solely from water and not from hydrogen gas. Hydrogen gas served only as an electron source in methanogenesis. A whole-cell product isotope discrimination of 1.5 favoring hydrogen over deuterium was observed in methane production in 81 atom% deuterated water. The distribution of deuterated methane species is described by a simple model of the overall reaction.