Total OH reactivity measurements at the biosphere-atmosphere interface

The biosphere emits copiously volatile organic compounds (VOCs) into the atmosphere, which are removed again depending on the oxidative capacity of the atmosphere and physical processes such as mixing, transport and deposition. Biogenic VOCs react with the primary oxidant of the atmosphere, the hydroxyl radical (OH), and potentially lead to the formation tropospheric ozone and aerosol, which impact regional climate and air quality. The rate of OH decay in the atmosphere, the total OH reactivity is a function of the atmospheric, reactive compound's concentration and reaction velocity with OH. One way to measure the total OH reactivity, the total OH sink, is with the Comparative Reactivity Method - CRM. Basically, the reaction of OH with a reagent (here pyrrole) in clean air and in the presence of atmospheric, reactive molecules is compared. This thesis presents measurements of the total OH reactivity at the biosphere-atmosphere interface to analyze various influences and driving forces. For measurements in natural environment the instrument was automated and a direct, undisturbed sampling method developed. Additionally, an alternative detection system was tested and compared to the originally used detector (Proton Transfer Reaction-Mass Spectrometer, PTR-MS). The GC-PID (Gas Chromatographic Photo-Ionization Detector) was found as a smaller, less expensive, and robust alternative for total OH reactivity measurements. The HUMPPA-COPEC 2010 measurement campaign in the Finish forest was impacted by normal boreal forest emissions as well as prolonged heat and biomass burning emissions. The measurement of total OH reactivity was compared with a comprehensive set of monitored individual species ambient concentration levels. A significant discrepancy between those individually measured OH sinks and the total OH reactivity was observed, which was characterized in detail by the comparison of within and above the forest canopy detected OH reactivity. Direct impact of biogenic emissions on total OH reactivity was examined on Kleiner Feldberg, Germany, 2011. Trans-seasonal measurements of an enclosed Norway spruce branch were conducted via PTR-MS, for individual compound's emission rates, and CRM, for total OH reactivity emission fluxes. Especially during summertime, the individually monitored OH sink terms could not account for the measured total OH reactivity. A controlled oxidation experiment in a low NOx environment was conducted in the EUPHORE reaction chamber (CHEERS, Spain 2011). The concentration levels of the reactant isoprene and its major products were monitored and compared to total OH reactivity measurements as well as to the results of two models. The individually measured compounds could account for the total OH reactivity during this experiment as well as the traditional model-degradation scheme for isoprene (MCM 3.2). Due to previous observations of high OH levels in the isoprene-rich environment of the tropics, a novel isoprene mechanism was recently suggested. In this mechanism (MIME v4) additional OH is generated during isoprene oxidation, which could not be verified in the conditions of the CHEERS experiment.

Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

Protein-protein interaction between phototaxis receptor sensory Rhodopsin I and its transducer HtrI

The molecular complex containing the seven transmembrane helix photoreceptor S&barbelow;ensory R&barbelow;hodopsin I&barbelow; (SRI) and transducer protein HtrI (H&barbelow;alobacterial Transducer for SRI&barbelow;) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) a repellent response by a two-photon reaction. Three aspects of SRI-HtrI structure/function and the signal transduction pathway were explored. First, the coupling of HtrI to the photoactive site of SRI was analyzed by mutagenesis and kinetic spectroscopy. Second, SRI-HtrI mutations and suppressors were selected and characterized to elucidate the color-sensing mechanism. Third, the signal relay through the transducer-bound histidine kinase was analyzed using an in vitro reconstitution system with known and newly identified taxis components. ^ Twenty-one mutations on HtrI were introduced by site-directed mutagenesis. Several replacements of charged residues perturbed the photochemical kinetics of SRI which led to the finding of a cluster of residues at the membrane/cytoplasm interface in HtrI electrostatically coupled to the photoactive site of SRI. We found by laser-flash kinetic spectroscopy that the transducer and these residues have specific effects on the light-induced proton transfer between the retinal chromophore and the protein. ^ One of the mutations showed an unusual mutant phenotype we called “inverted” signaling, in which the cell produces a repellent response to normally attractant light. Therefore, this mutant (E56Q of HtrI) had lost the color-discrimination by the SRI-HtrI complex. We used suppressor analysis to better understand the phenotype. Certain suppressors resulted in return of attractant responses to orange light but with inversion of the normally repellent response to white light to an attractant response. To explain this and other results, we formulated the Conformational Shuttling model in which the HtrI-SRI complex is poised in a metastable equilibrium of two conformations shifted in opposite directions by orange and white light. We tested this model by behavioral analysis (computerized cell tracking and motion study) of double mutants of inverting and suppressing mutations and the results confirmed the equilibrium-shift explanation. ^ We developed an in vitro system for measuring the effect of purified transducer on the histidine-kinase CheAH that controls the flagellar motor switch. The rate of kinase autophosphorylation was stimulated >2 fold in the reconstitution of the complete signal transduction system from purified components from H. salinarum. The in vitro assay also showed that the kinase activity was reduced in the absence and in the presence of high levels of linker protein CheWH. (Abstract shortened by UMI.) ^

Effects of sources and meteorology on particulate matter in the Western Mediterranean Basin: An overview of the DAURE campaign

DAURE (Determination of the Sources of Atmospheric Aerosols in Urban and Rural Environments in the Western Mediterranean) was a multidisciplinary international field campaign aimed at investigating the sources and meteorological controls of particulate matter in the Western Mediterranean Basin (WMB). Measurements were simultaneously performed at an urban-coastal (Barcelona, BCN) and a rural-elevated (Montseny, MSY) site pair in NE Spain during winter and summer. State-of-the-art methods such as 14C analysis, proton-transfer reaction mass spectrometry, and high-resolution aerosol mass spectrometry were applied for the first time in the WMB as part of DAURE. WMB regional pollution episodes were associated with high concentrations of inorganic and organic species formed during the transport to inland areas and built up at regional scales. Winter pollutants accumulation depended on the degree of regional stagnation of an air mass under anticyclonic conditions and the planetary boundary layer height. In summer, regional recirculation and biogenic secondary organic aerosols (SOA) formation mainly determined the regional pollutant concentrations. The contribution from fossil sources to organic carbon (OC) and elemental carbon (EC) and hydrocarbon-like organic aerosol concentrations were higher at BCN compared with MSY due to traffic emissions. The relative contribution of nonfossil OC was higher at MSY especially in summer due to biogenic emissions. The fossil OC/EC ratio at MSY was twice the corresponding ratio at BCN indicating that a substantial fraction of fossil OC was due to fossil SOA. In winter, BCN cooking emissions were identified as an important source of modern carbon in primary organic aerosol.

Mutation of a single residue in the ba 3 oxidase specifically impairs protonation of the pump site

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

Qualitative Distinction of Autotrophic and Heterotrophic Processes at the Leaf Level by Means of Triple Stable Isotope (C–O–H) Patterns

Foliar samples were harvested from two oaks, a beech, and a yew at the same site in order to trace the development of the leaves over an entire vegetation season. Cellulose yield and stable isotopic compositions (δ13C, δ18O, and δD) were analyzed on leaf cellulose. All parameters unequivocally define a juvenile and a mature period in the foliar expansion of each species. The accompanying shifts of the δ13C-values are in agreement with the transition from remobilized carbohydrates (juvenile period), to current photosynthates (mature phase). While the opponent seasonal trends of δ18O of blade and vein cellulose are in perfect agreement with the state-of-art mechanistic understanding, the lack of this discrepancy for δD, documented for the first time, is unexpected. For example, the offset range of 18 permil (oak veins) to 57 permil (oak blades) in δD may represent a process driven shift from autotrophic to heterotrophic processes. The shared pattern between blade and vein found for both oak and beech suggests an overwhelming metabolic isotope effect on δD that might be accompanied by proton transfer linked to the Calvin-cycle. These results provide strong evidence that hydrogen and oxygen are under different biochemical controls even at the leaf level.

Highly stereoselective decarboxylation of (+)-1-Bromo-1-chloro-2,2,2-trifluoropropanoic acid gives (+)-1-Bromo-1-chloro-2,2,2-trifluoroethane ((+)-Halothane) with retention of configuration

The absolute configuration of the title acid (2) has been determined to be S by X-ray crystallography. Thus, decarboxylation of 2 produces (S)-(+)-halothane with 99% retention of configuration. This behavior is compared to other stereoselective decarboxylation reactions of ?-haloacids from the literature that also give high degrees of retention of configuration when in the form of their quaternary ammonium salts, which contain one proton. The proton of the ammonium salt is necessary to protonate the anionic intermediate formed from decarboxylation. In the absence of this relatively acidic proton, we had previously found that using triethylene glycol (TEG) as both solvent and proton source for the decarboxylation reaction of acid 2 caused poor stereoselectivity. This was in contrast to 1,2,2,2-tetrafluoro-1-methoxypropionic acid (6), which showed a high degree of retention of configuration in TEG. To rationalize this differing behavior we report DFT studies at PCM-B3LYP/6-31++G** level of theory (the results were additionally confirmed with 6-311++G** and aug-cc-pVDZ basis sets). The energy barrier to inversion of configuration of the anionic reaction intermediate of acid 2 (11) is 10.23 kcal/mol. However, we find that the anionic intermediate from acid 6 (10) would rather undergo ?-elimination instead of inversion of configuration. Thus the planar transition state required for inversion of configuration is never reached, regardless of the rate of proton transfer to the anion.

Efficient exchange of the primary quinone acceptor QA in isolated reaction centers of Rhodopseudomonas viridis

A key step in the conversion of solar energy into chemical energy by photosynthetic reaction centers (RCs) occurs at the level of the two quinones, QA and QB, where electron transfer couples to proton transfer. A great deal of our understanding of the mechanisms of these coupled reactions relies on the seminal work of Okamura et al. [Okamura, M. Y., Isaacson, R. A., & Feher, G. (1975) Proc. Natl. Acad. Sci. USA 88, 3491–3495], who were able to extract with detergents the firmly bound ubiquinone QA from the RC of Rhodobacter sphaeroides and reconstitute the site with extraneous quinones. Up to now a comparable protocol was lacking for the RC of Rhodopseudomonas viridis despite the fact that its QA site, which contains 2-methyl-3-nonaprenyl-1,4-naphthoquinone (menaquinone-9), has provided the best x-ray structure available. Fourier transform infrared difference spectroscopy, together with the use of isotopically labeled quinones, can probe the interaction of QA with the RC protein. We establish that a simple incubation procedure of isolated RCs of Rp. viridis with an excess of extraneous quinone allows the menaquinone-9 in the QA site to be almost quantitatively replaced either by vitamin K1, a close analogue of menaquinone-9, or by ubiquinone. To our knowledge, this is the first report of quinone exchange in bacterial photosynthesis. The Fourier transform infrared data on the quinone and semiquinone vibrations show a close similarity in the bonding interactions of vitamin K1 with the protein at the QA site of Rp. viridis and Rb. sphaeroides, whereas for ubiquinone these interactions are significantly different. The results are interpreted in terms of slightly inequivalent quinone–protein interactions by comparison with the crystallographic data available for the QA site of the two RCs.

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 μs at pH 7 to 45 μs at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 μs and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

Tight Asp-85–Thr-89 association during the pump switch of bacteriorhodopsin

Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O—H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3-18O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O—D group of Thr-89 in D2O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation of this hydrogen bond remains unchanged in the L intermediate and through the M intermediate. Furthermore, a strong interaction between Asp-85 and the O—H (O—D) group of Thr-89 in M is indicated by a shift in the C⩵O stretching vibration of the former because of 18O substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M, contrary to structural models based on x-ray crystallography of the photocycle intermediates. We propose that, upon photoisomerization of the chromophore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switching process. On the other hand, the loss of hydrogen bonding at the other oxygen of Asp-85 in M may be related to the switching event.

Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-Å resolution

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-Å resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an ɛ-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxido-reductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at 1.8-A resolution. In the sulfo-form the molybdenum molybdopterin cytosine dinucleotide cofactor has a dithiolene-bound fac-[Mo, = O, = S, ---(OH2)] substructure. Bound inhibitory isopropanol in the inner compartment of the substrate binding tunnel is a model for the Michaelis complex of the reaction with aldehydes (H-C = O,-R). The reaction is proposed to proceed by transfer of the molybdenum-bound water molecule as OH- after proton transfer to Glu-869 to the carbonyl carbon of the substrate in concert with hydride transfer to the sulfido group to generate [MoIV, = O, -SH, ---(O-C = O, -R)). Dissociation of the carboxylic acid product may be facilitated by transient binding of Glu-869 to the molybdenum. The metal-bound water is replenished from a chain of internal water molecules. A second alcohol binding site in the spacious outer compartment may cause the strong substrate inhibition observed. This compartment is the putative binding site of large inhibitors of xanthine oxidase.

NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 10(10). Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4 beta-proton to the 6 beta-position, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pKa values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.

Progress toward chemical accuracy in the computer simulation of condensed phase reactions.

We describe a procedure for the generation of chemically accurate computer-simulation models to study chemical reactions in the condensed phase. The process involves (i) the use of a coupled semiempirical quantum and classical molecular mechanics method to represent solutes and solvent, respectively; (ii) the optimization of semiempirical quantum mechanics (QM) parameters to produce a computationally efficient and chemically accurate QM model; (iii) the calibration of a quantum/classical microsolvation model using ab initio quantum theory; and (iv) the use of statistical mechanical principles and methods to simulate, on massively parallel computers, the thermodynamic properties of chemical reactions in aqueous solution. The utility of this process is demonstrated by the calculation of the enthalpy of reaction in vacuum and free energy change in aqueous solution for a proton transfer involving methanol, methoxide, imidazole, and imidazolium, which are functional groups involved with proton transfers in many biochemical systems. An optimized semiempirical QM model is produced, which results in the calculation of heats of formation of the above chemical species to within 1.0 kcal/mol (1 kcal = 4.18 kJ) of experimental values. The use of the calibrated QM and microsolvation QM/MM (molecular mechanics) models for the simulation of a proton transfer in aqueous solution gives a calculated free energy that is within 1.0 kcal/mol (12.2 calculated vs. 12.8 experimental) of a value estimated from experimental pKa values of the reacting species.

Conformations and folding of lysozyme ions in vacuo.

Proton transfer reactivity of isolated charge states of the protein hen egg-white lysozyme shows that multiple distinct conformations of this protein are stable in the gas phase. The reactivities of the 9+ and 10+ charge state ions, formed by electrospray ionization of "native" (disulfide-intact) and "denatured" (disulfide-reduced) solutions, are consistent with values calculated for ions in their crystal structure and fully denatured conformations, respectively. Charge states below 8+ of both forms, formed by proton stripping, have similar or indistinguishable reactivities, indicating that the disulfide-reduced ions fold in the gas phase to a more compact conformation.