Studies on the reactivity of malondialdehyde. I. Effects of acid concentration and solvent composition on deuterium exchange reactions

The rates of deuterium exchange reactions of malondialdehyde (MDA) and deuterated malondialdehyde (MDAd) have been studied as a function of acidity and the content of dimethyl sulfoxide (DMSO) in binary mixtures with D₂O . MDA incorporates deuterium from D₂O solutions in a first-order reaction with a rate constant (k_obs) that depends on the acid concentration. From this dependence, a catalytic constant, k_cat, can be derived (k_cat^MDA = 2.25 × 10⁵M^?s^?1). Similar kinetic behavior was found for MDAd in H₂O solutions, and in this case, k_cat^MDA = 1.56 × 10⁵M^?1s^?1. Results from reactions of MDA and MDAd in identical H₂OD₂O mixtures show that primary and secondary isotope effects are small (k_H/k_D = 1.13) and that solvent isotope effects cause most of the differences found between reactions in D₂O and H₂O. Reactions in binary DMSOd₆D₂O mixtures show a six-fold rate increase as the proportion of DMSOd₆ increases from 50% to 90%. These results also illustrate the relatively high reactivity of MDA at pH values well above its pK_a and the importance of medium composition on its reaction rate.     

Intraspecific variation in hair delta(13)C and delta(15)N values of ring-tailed lemurs (Lemur catta) with known individual histories, behavior, and feeding ecology

Stable carbon and nitrogen isotope compositions were analyzed from hair samples of 30 sympatric ring-tailed lemurs (Lemur catta) inhabiting the Beza Mahafaly Special Reserve, Madagascar. All lemurs were known individuals involved in a longitudinal study, which allowed us to explore the degree to which group membership, sex, health status, and migration influenced their stable isotope compositions. The differences in delta(13)C and delta(15)N values between groups were small (<1.5 per thousand) but highly significant. In fact, each group was tightly clustered, and discriminant function analysis of the stable isotope data assigned individuals to the group in which they were originally collared with over 90% accuracy. In general, the differences between groups reflected the degree to which they utilized forested versus open habitats. As open habitats at Beza Mahafaly often correspond to areas of anthropogenic disturbance, these data suggest that isotopic data can be useful for addressing questions of lemur conservation. There were few sex differences, but significant differences did occur between individuals of normal and suboptimal health, with those in poor health (especially those in the worst condition) being enriched in (15)N and to a lesser degree (13)C compared with healthy individuals. Moreover, lemurs that had emigrated between 2003 and 2004 had different delta(13)C and delta(15)N compositions than their original groups.     

Influence of water flow on skeletal isotopic composition in the coral Pocillopora damicornis

Fragments of branching Pocillopora damicornis coral colonies were grown in experimental flumes under two water flow regimes. Colony size and buoyant weight increased most rapidly in the fast-flow regime. Branch tips from the upper and outer parts of the colonies showed the lowest and most consistent skeletal oxygen isotope ratios. Flow regime had little influence on the lowest oxygen isotope ratios, which were at least 3.5‰ lighter than the calculated oxygen isotopic equilibrium. These “kinetic” isotope effects are comparable to those observed in Porites corals. Relatively more branch tips showed extreme ¹⁸O depletions under low-flow conditions, and among small coral colonies. Isotopic variability was greater among branch tips from the lower and inner parts of the colonies and at high flow. Skeletal oxygen and carbon isotope ratios generally showed positive correlations. Despite the particularly large offsets from isotopic equilibrium, the isotopically lightest branches showed the greatest isotopic consistency and therefore would make the best isotopic thermometers. Isotopic variability within the colony may provide an indication of flow regime.     

An investigation into the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine using heavy atom kinetic isotope effects

Heavy atom kinetic isotope effects (KIEs) were determined for the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine (FTC). The leaving-S, carbonyl-C, and carbonyl-O KIEs are ³⁴k = 0.994 ± 0.004, ¹³k = 1.0148 ± 0.0007, and ¹⁸k = 0.999 ± 0.002, respectively. The observed KIEs support a mechanism for both acylation and deacylation where the steps up to and including the formation of the tetrahedral intermediate are at least partially rate determining. These results, in contrast to previous studies with acetylthiocholine, suggest that the decomposition of a tetrahedral intermediate is not rate-determining for FTC hydrolysis. Structural differences between the two substrates are likely responsible for the observed mechanism change with FTC.     

Editing of Multidimensional NMR Spectra of Partially Deuterated Proteins. Measurement of Amide Deuterium Isotope Effects on the Chemical Shifts of Protein Backbone Nuclei

Novel multidimensional NMR pulse sequences are presented for determination of amide deuterium isotope effects on the 13C chemical shifts of the backbone in proteins. The sequences edit heteronuclear triple resonance spectra into two subspectra according to whether a deuterium or a proton is attached to 15N for the pertinent correlations. The new experiments are demonstrated using 13C, 15N-labeled RAP 17-112 (N-terminal domain of 2-macroglobulin receptor associated protein).     

Enrichment and characterization of ammonia-oxidizing archaea from the open ocean: phylogeny,physiology and stable isotope fractionation

Archaeal genes for ammonia oxidation are widespread in the marine environment, but direct physiological evidence for ammonia oxidation by marine archaea is limited. We report the enrichment and characterization of three strains of pelagic ammonia-oxidizing archaea (AOA) from the North Pacific Ocean that have been maintained in laboratory culture for over 3 years. Phylogenetic analyses indicate the three strains belong to a previously identified clade of water column-associated AOA and possess 16S ribosomal RNA genes and ammonia monooxygenase subunit a (amoA) genes highly similar (98–99% identity) to those recovered in DNA and complementary DNA clone libraries from the open ocean. The strains grow in natural seawater-based liquid medium while stoichiometrically converting ammonia (NH₃) to nitrite (NO₂⁻). Ammonia oxidation by the enrichments is only partially inhibited by allylthiourea at concentrations known to completely inhibit cultivated ammonia-oxidizing bacteria. The three strains were used to determine the nitrogen stable isotope effect (¹⁵ɛ_NH3) during archaeal ammonia oxidation, an important parameter for interpreting stable isotope ratios in the environment. Archaeal ¹⁵ɛ_NH3 ranged from 13‰ to 41‰, within the range of that previously reported for ammonia-oxidizing bacteria. Despite low amino acid identity between the archaeal and bacterial Amo proteins, their functional diversity as captured by ¹⁵ɛ_NH3 is similar.     

Can heavy isotopes increase lifespan? Studies of relative abundance in various organisms reveal chemical perspectives on aging

Stable heavy isotopes co‐exist with their lighter counterparts in all elements commonly found in biology. These heavy isotopes represent a low natural abundance in isotopic composition but impose great retardation effects in chemical reactions because of kinetic isotopic effects (KIEs). Previous isotope analyses have recorded pervasive enrichment or depletion of heavy isotopes in various organisms, strongly supporting the capability of biological systems to distinguish different isotopes. This capability has recently been found to lead to general decline of heavy isotopes in metabolites during yeast aging. Conversely, supplementing heavy isotopes in growth medium promotes longevity. Whether this observation prevails in other organisms is not known, but it potentially bears promise in promoting human longevity.     

Interpretation of V/K isotope effects for enzymatic reactions exhibiting multiple isotopically sensitive steps

Formulations of an enzyme mechanism where only a single step is presumed to be isotopically sensitive can be written in terms of forward and reverse commitments to catalysis. These commitments provide a natural and intuitive way of interpreting the observed isotope effects. Unfortunately, when multiple isotopically sensitive steps are present in the mechanism, including effects associated with pre-equilibria of the unbound substrate, the observed V/K kinetic isotope effect is expressed as a complicated expression of the intrinsic rate constants for each step, the interpretation of which is not always immediately obvious. We show here that V/K isotope effects from unbranched or rapid-equilibrium random Michaelis-Menten systems containing multiple isotopically sensitive steps can be written as a weighted average of the intrinsic isotope effects on each step, where this intrinsic isotope effect from each step is the product of the equilibrium isotope effect on the formation of the reacting intermediate for that step and the intrinsic kinetic effect on the forward rate constant for that step, and the weighting factors are simply the reciprocal sum of the forward and reverse commitments for each step i plus unity, 1/(C(fi)+C(ri)+1), equivalent to the sensitivity index [Ray, W.J., 1983.     

Broønsted analysis of aspartate aminotransferase via exogenous catalysis of reactions of an inactive mutant

Primary amines functionally replace lysine 258 by catalyzing both the 1,3-prototropic shift and external aldimine hydrolysis reactions with the inactive aspartate aminotransferase mutant K258A. This finding allows classical Brønsted analyses of proton transfer reactions to be applied to enzyme-catalyzed reactions. An earlier study of the reaction of K258A with cysteine sulfinate (Toney, M.D. & Kirsch, J.F., 1989, Science 243, 1485) provided a beta value of 0.4 for the 1,3-prototropic shift. The beta value reported here for the transamination of oxalacetate to aspartate is 0.6. The catalytic efficacy of primary amines is largely determined by basicity and molecular volume. The dependence of the rate constants for the reactions of K258A and K258M on amine molecular volume is nearly identical. This observation argues that the alkyl groups of the added amines do not occupy the position of the lysine 258 side chain in the wild type enzyme. Large primary C alpha and insignificant solvent deuterium kinetic isotope effects with amino acid substrates demonstrate that the amine nitrogen of the exogenous catalysts directly abstracts the labile proton in the rate-determining step.     

Mechanistic and structural aspects of photosynthetic water oxidation

Conclusions on the functional and structural organisation of photosynthetic water oxidation are gathered from a critical survey of the wealth of data reported in the literature and author's own experimental research: (1) the water oxidising complex (WOC) contains a tetranuclear manganese cluster of dimer of dimers' structure and functional heterogeneity of the metal centers, (2) the four step univalent oxidative pathway leading to water oxidation into molecular oxygen and four protons comprises only manganese, tyrosine Y_Z of polypeptide Dl and the substrate as redox active species, (3) the redox transitions S₀→ S₁ and S₁→ S₂ are manganese centered whereas S₂→ S₃ is most likely a ligand-centered reaction, (4) there exist several lines of evidence for a marked structural change that accompanies the redox transition S₂→ S₃, (5) one Ca²⁺ is an indispensible constituent of a functionally competent WOC while the role of Cl is much less clear and a direct participation disputable, (6) substrate water is most likely bound in all redox states S₀,…,S₃ and exchangeable with the bulk phase. The protonation state is determined by the redox state S₁ and the protein microenvironment. A mechanism is proposed for water oxidation in the WOC that is based on three key postulates: (1) water oxidation takes place in the first coordination sphere of one manganese dimer [Mn_aMn_b]; (2) the essential O-O bond is preformed in S₃ as part of a rapid redox isomerism S₃(I)→S₃(II) where in S₃(II) a nuclear geometry and electronic configuration is attained that corresponds to a peroxidic-type species; and (3) S₃(II) is an ‘entatic state’ for the formation of complexed dioxygen triggered by Y_Z^OX induced electron abstraction from the WOC and electronic redistribution to S₀(O₂).     

Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, deltaH(o), deltaS(o), and deltaC(o)p and evaluation of solvent isotope effects.

The stability of the N-terminal domain of the ribosomal protein L9, NTL9, from Bacillus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, deltaH(o), deltaS(o), and deltaC(o)p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs-Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3.78 +/- 0.25 kcal mol(-1) at 14 degrees C. DeltaH(o)(25 degrees C) for protein unfolding equals 9.9 +/- 0.7 kcal mol(-1) and TdeltaS(o)++(25 degrees C) equals 6.2 +/- 0.6 kcal mol(-1). DeltaC(o)p equals 0.53 +/- 0.06 kcal mol(-1) deg(-1). There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1.06 +/- 0.60 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 5.8 +/- 3.6 degrees C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0.68 +/- 0.68 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 3.5 +/- 2.1 degrees C in D2O. Within experimental error there are no changes in deltaH(o) (25 degrees C) when D2O is substituted for H2O.     

Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H₂O/50%D₂O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a ¹³C′-coupled 2D ¹⁵N–¹H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH₂ groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis

Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented.     

Oxidative dechlorination of halogenated phenols catalyzed by two distinct enzymes: Horseradish peroxidase and dehaloperoxidase

The mechanism of the dehalogenation step catalyzed by dehaloperoxidase (DHP) from Amphitrite ornata, an unusual heme-containing protein with a globin fold and peroxidase activity, has remarkable similarity with that of the classical heme peroxidase, horseradish peroxidase (HRP). Based on quantum mechanical/molecular mechanical (QM/MM) modeling and experimentally determined chlorine kinetic isotope effects, we have concluded that two sequential one electron oxidations of the halogenated phenol substrate leads to a cationic intermediate that strongly resembles a Meisenheimer intermediate – a commonly formed reactive complex during nucleophilic aromatic substitution reactions especially in the case of arenes carrying electron withdrawing groups.     

Investigating the biochar effects on C‐mineralization and sequestration of carbon in soil compared with conventional amendments using the stable isotope (δ13C) approach

Biomass‐derived black carbon (biochar) is considered to be an effective tool to mitigate global warming by long‐term C‐sequestration in soil and to influence C‐mineralization via priming effects. However, the underlying mechanism of biochar (BC) priming relative to conventional biowaste (BW) amendments remains uncertain. Here, we used a stable carbon isotope (δ¹³C) approach to estimate the possible biochar effects on native soil C‐mineralization compared with various BW additions and potential carbon sequestration. The results show that immediately after application, BC suppresses and then increases C‐mineralization, causing a loss of 0.14–7.17 mg‐CO₂–C g^?1‐C compared to the control (0.24–1.86 mg‐CO₂–C g^?1‐C) over 1–120 days. Negative priming was observed for BC compared to various BW amendments (?10.22 to ?23.56 mg‐CO₂–C g^?1‐soil‐C); however, it was trivially positive relative to that of the control (8.64 mg‐CO₂–C g^?1‐soil‐C). Furthermore, according to the residual carbon and δ¹³C signature of postexperimental soil carbon, BC‐C significantly increased (P < 0.05) the soil carbon stock by carbon sequestration in soil compared with various biowaste amendments. The results of cumulative CO₂–C emissions, relative priming effects, and carbon storage indicate that BC reduces C‐mineralization, resulting in greater C‐sequestration compared with other BW amendments, and the magnitude of this effect initially increases and then decreases and stabilizes over time, possibly due to the presence of recalcitrant‐C (4.92 mg‐C g^?1‐soil) in BC, the reduced microbial activity, and the sorption of labile organic carbon (OC) onto BC particles.     

The consequence of species loss on ecosystem nitrogen cycling depends on community compensation

Repercussions of species loss on ecosystem processes depend on the effects of the lost species as well as the compensatory responses of the remaining species in the community. We experimentally removed two co-dominant plant species and added a ¹⁵N tracer in alpine tundra to compare how species’ functional differences influence community structure and N cycling. For both of the species, production compensated for the biomass removed by the second year. However, the responses of the remaining species depended on which species was removed. These differences in compensation influenced how species loss impacted ecosystem processes. After the removal of one of the co-dominant species, Acomastylis rossii, there were few changes in the relative abundance of the remaining species, and differences in functioning could be predicted based on effects associated with the removed species. In contrast, the removal of the other co-dominant, Deschampsia caespitosa, was associated with subsequent changes in community structure (species relative abundances and diversity) and impacts on ecosystem properties (microbial biomass N, dissolved organic N, and N uptake of subordinate species). Variation in compensation may contribute to the resulting effects on ecosystem functioning, with the potential to buffer or accelerate the effects of species loss.     

Stable Isotopes and Carbon Cycle Processes in Forests and Grasslands

Abstract: Scaling and partitioning are frequently two difficult challenges facing ecology today. With regard to ecosystem carbon balance studies, ecologists and atmospheric scientists are often interested in asking how fluxes of carbon dioxide scale across the landscape, region and continent. Yet at the same time, physiological ecologists and ecosystem ecologists are interested in dissecting the net ecosystem CO₂ exchange between the biosphere and the atmosphere to achieve a better understanding of the balance between photosynthesis and respiration within a forest. In both of these multiple-scale ecological questions, stable isotope analyses of carbon dioxide can play a central role in influencing our understanding of the extent to which terrestrial ecosystems are carbon sinks. In this synthesis, we review the theory and present field evidence to address isotopic scaling of CO₂ fluxes. We first show that the ¹³C isotopic signal which ecosystems impart to the atmosphere does not remain constant over time at either temporal or spatial scales. The relative balances of different biological activities and plant responses to stress result in dynamic changes in the ¹³C isotopic exchange between the biosphere and atmosphere, with both seasonal and stand-age factors playing major roles influencing the ¹³C biosphere-atmosphere exchange. We then examine how stable isotopes are used to partition net ecosystem exchange fluxes in order to calculate shifts in the balance of photosynthesis and respiration. Lastly, we explore how fundamental differences in the ¹⁸O isotopic gas exchange of forest and grassland ecosystems can be used to further partition terrestrial fluxes.     

Physical and chemical basis of carbon isotope fractionation in plants

Naturally-occurring variations in the abundances of the stable isotopes of carbon and other elements can be used to understand the dynamics of natural processes in chemistry, biochemistry, biology, medicine, ecology and other fields. The use of carbon-13 isotopic abundances as an indicator of photosynthetic function in plants has become common. The purpose of this article is to describe the physical and chemical processes that contribute to the abundances of carbon-13 in plant materials, and to provide a framework for understanding how those processes control the isotopic contents of natural materials.