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Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to –340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°’ = −493 mV. Here we demonstrate by X-ray structural analyses, QM/MM computational studies, and multiple spectroscopy/activity based titrations that highly cooperative electron transfer (n = 3) from a low-potential one-electron (FAD) to a two-electron (FMN) transferring flavin cofactor is the key to overcome the resonance stabilized aromatic system by hydride transfer in a highly hydrophobic pocket. The results evidence how the protein environment inversely functionalizes two flavins to switch from low-potential one-electron to hydride transfer at the thermodynamic limit of flavin redox chemistry.
Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination
Experimental results are presented for 180 in silico designed octapeptide sequences and their stabilizing effects on the major histocompatibility class I molecule H-2Kb. Peptide sequence design was accomplished by a combination of an ant colony optimization algorithm with artificial neural network classifiers. Experimental tests yielded nine H-2Kb stabilizing and 171 nonstabilizing peptides. 28 among the nonstabilizing octapeptides contain canonical motif residues known to be favorable for MHC I stabilization. For characterization of the area covered by stabilizing and non-stabilizing octapeptides in sequence space, we visualized the distribution of 100,603 octapeptides using a self-organizing map. The experimental results present evidence that the canonical sequence motives of the SYFPEITHI database on their own are insufficient for predicting MHC I protein stabilization.
To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase. KM and kcat values and the bimolecular rate constant were determined under steady- or presteady-state conditions, respectively. We present evidence that Trp-121 which is surrounded by a hydrophobic patch is the electron entry site to oxidase. Mutations in this cluster do not affect the binding of cytochrome c as the KM remains largely unchanged. Rather, the kcat is reduced, proposing that these hydrophobic residues are required for a fine tuning of the redox partners in the initial collisional complex to obtain a configuration optimal for electron transfer.
Photoelectron (PE) spectra of ethylene and vinylene carbonates and thiocarbonates as well as of methylene trithiocarbonate and some open-chain derivatives are reported.
The low energy bands, well separated in the unsaturated compounds, are assigned to lone pair and π type ionizations. The assignment is based on comparison of PE spectra, modified CNDO calculations, and sulfur Κβ emission spectra. The pronounced substituent effects due to which the first ionization potential varies from 8.4 eV to 11.1 eV are discussed.
Sulfhydryl Groups, Methylmercury Containing Inactivator, Coenzyme Analogue Nicotinamide-(S-methylmercury-thioinosine) dinucleotide was formed by reaction of nicotin amide-(6-thiopurine) dinucleotide with methylmercury chloride. The compound exhibits coenzyme properties in the test with LDH (Km=1.5 × 10-4 м , Vmax=12500) and LADH (Km=1.7 × 10-4 м, Vmax=27) and inactivates YADH and GAPDH. From incubations with LDH and LADH the mercury containing coenzyme could be regained by column chromatography. The compound seems to be qualified for the X-ray structure analysis of the coenzyme-enzyme complex for some dehyrogenases based on the proportion of the heavy metal.
Lactate dehydrogenase from pig heart is inactivated by the NAD+ -analog P1-N6-(4-azidophenylethyl)adenosine-P2-[4-(3-azidopyridinio)butyl]diphosphate (6) upon irradiation with UV light of wavelengths in the range from 300 to 380 nm. The decrease in enzyme activity can be prevented by the addition of NAD+ and oxalate. The modified enzyme shows a reduced binding capacity for its coenzyme as compared to native lactate dehydrogenase. The amount of incorporated coenzyme is deduced from the ribose content of inactivated enzyme. Tryptic digestion of the modified protein and separation of the peptides by HPLC yields 5 ribose-containing fractions. One of them, fraction 6 6 , is split by treatment with nucleotide pyrophosphatase into two subfractions, 63 and 58. Only subfraction 63 contains ribose. Whereas peptide 58 shows a UV absorption spectrum similar to that of 4-(3-aminopyridinio)-butyl phosphate (3). Amino acid analyses of the peptides indicate that the inactivator forms covalent bonds with different parts of the protein: Peptide 63 is characterized by a great portion of hydrophobic amino acids whereas peptide 58 shows a high degree of hydrophilicity.
The transporter associated with antigen processing (TAP)-like (TAPL, ABCB9) belongs to the ATP-binding cassette transporter family, which translocates a vast variety of solutes across membranes. The function of this half-size transporter has not yet been determined. Here, we show that TAPL forms a homodimeric complex, which translocates peptides across the membrane. Peptide transport strictly requires ATP hydrolysis. The transport follows Michaelis-Menten kinetics with low affinity and high capacity. Different nucleotides bind and energize the transport with a slight predilection for purine bases. The peptide specificity is very broad, ranging from 6-mer up to at least 59-mer peptides with a preference for 23-mers. Peptides are recognized via their backbone, including the free N and C termini as well as side chain interactions. Although related to TAP, TAPL is unique as far as its interaction partners, transport properties, and substrate specificities are concerned, thus excluding that TAPL is part of the peptide-loading complex in the classic route of antigen processing via major histocompatibility complex class I molecules.
Photo-initiated processes, like photo-excitation and -deexcitation, internal conversion, excitation energy transfer and electron transfer, are of importance in many areas of physics, chemistry and biology. For the understanding of such processes, detailed knowledge of excitation energies, potential energy surfaces and excited state properties of the involved molecules is an essential prerequisite. To obtain these informations, quantum chemical calculations are required. Several quantum chemical methods exist which allow for the calculation of excited states. Most of these methods are computationally costly what makes them only applicable to small molecules. However, many biological systems where photo-processes are of interest like light-harvesting complexes in photosynthesis or the reception of light in the human eye by rhodopsin are quite large. For large systems, however, only few theoretical methods remain applicable. The currently most widely used method is time-dependent density functional theory (TD-DFT), which can treat systems of up to 200–300 atoms with the excitation energies of some excited states exhibiting errors of less than 0.5 eV. Yet, TD-DFT has several drawbacks. The most severe failure of TD-DFT is the false description of charge transfer states which is particularly problematic in case of larger systems where it yields a multitude of artificially low-lying charge transfer states. But also Rydberg states and states with large double excitation character are not described correctly. Still, if these deficiencies are kept in mind during the interpretation of results, TD-DFT is a useful tool for the calculation of excited states. In my thesis, TD-DFT is applied in investigations of excitation energy and electron transfer processes in light-harvesting complexes. Since light-harvesting complexes, which consist of thousands of atoms, are by far too large to be calculated, model complexes for the processes of interest are constructed from available crystal structures. The model complexes are used to calculate potential energy curves along meaningful reaction coordinates. Artificial charge transfer states are corrected with the help of the so-called ∆DFT method. The resulting potential energy curves are then interpreted by comparison with experimental results. For the light-harvesting complex LH2 from purple bacteria the experimentally observed formation of carotenoid radical cations is studied. It is shown that the carotenoid radical cation is formed most likely via the optically forbidden S1 state of the carotenoid. In light-harvesting complex LHC-II of green plants the fast component of the so-called non-photochemical quenching (NPQ) is investigated. Two of several different hypotheses on the mechanism of NPQ, which have been proposed recently, are studied in detail. The first one suggests that NPQ proceeds via simple replacement of violaxanthin by zeaxanthin in the binding pocket in LHC-II. However, the calculated potential energy curves exhibit no difference between violaxanthin and zeaxanthin in the binding pocket. In combination with experimental results it is thus shown that simple replacement alone does not mediate NPQ in LHC-II. The second hypothesis proposes conformational changes of LHC-II that lead to quenching at the central lutein and chlorophyll molecules during NPQ. My TD-DFT calculations demonstrate that if this mechanism is operative, only the lutein 1 which is one of two central luteins present in LHC-II can take part in the quenching process. This is corroborated by recent experiments. Though several conclusions can be drawn from the investigations using TD-DFT, the interpretability of the results is limited due to the deficiencies of the method and of the models. To overcome the methodological deficiencies, more accurate methods have to be employed. Therefore, the so-called algebraic diagrammatic construction scheme (ADC) is implemented. ADC is a widely overlooked ab initio method for the calculation of excited states, which is based on propagator theory. Its theoretical derivation proceeds via perturbation expansion of the polarization propagator, which describes electronic excitations. This yields separate schemes for every order of perturbation theory. The second order scheme ADC(2), which is employed here, is the equivalent to the Møller-Plesset ground state method MP(2), but for excited states. It represents the computationally cheapest excited state method which can correctly describe doubly excited states, as well as Rydberg and charge transfer states. The quality of ADC(2) results is demonstrated in calculations on linear polyenes which serve as model systems for the larger carotenoid molecules. The calculations show that ADC(2) describes the three lowest excited states of polyenes sufficiently well, particularly the optically forbidden S1 state which is known to possess large double excitation character. Yet, the applicability of the method is limited compared to TD-DFT due to the much larger computational requirements. To facilitate the calculation of larger systems with ADC(2) a new variant of the method is developed and implemented. The variant employs the short-range behavior of electron correlation to reduce the computational effort. As a first step, the working equations of ADC(2) are transformed into a basis of local orbitals. In this basis negligible contributions of the equations which are due to electron correlation can be identified based on the distances of local orbitals. A so-called “bumping” scheme is implemented which removes the negligible parts during a calculation. This way, the computation times as well as the disk space requirements can be reduced. With the “bumping” scheme several new parameters are introduced that regulate the amount of “bumping” and thereby the speed and the accuracy of computations. To determine useful values for the parameters an evaluation is performed using the linear polyene octatetraene as test molecule. From the evaluation an optimal set of parameter values is obtained, so that the computation times become minimal, while the errors in the excitation energies due to the “bumping” do not exceed 0.15 eV. With further calculations on various molecules of different sizes it is tested if these parameter values are universal, i.e. if they can be used for all molecules. The test calculations show that the errors in the excitation energies are below 0.15 eV for all test systems. Additionally, no trend is visible for the errors that their magnitude might depend on the system. In contrast, the amount of disregarded contributions in the calculations increases drastically with growing system size. Thus, the local variant of ADC(2) can be used in future to reliably calculate excited states of systems which are not accessible with conventional ADC(2).
1. Fab co-complexes of proton pumping NADH:ubiquinone oxidoreductase (complex I) Fab fragments suitable for co-crystallization with complex I were generated using an immobilized papainbased protocol. The binding of the antibody fragments to complex I was verified using Surface Plasmon Resonance and size exclusion chromatography. The binding constants of the antibodies and their respective Fab fragments were found to be in the nanomolar range. This work presents the first report on successful crystallization of complex I (proton pumping NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica with proteolytic Fab fragments. The quality of the crystals was significantly improved when compared to the initial experiments and the best crystals diffracted X-rays to a resolution of ~7 Å. The activity of complex I remained uninfluenced by antibody fragment binding. The initial diffraction data suggest that the complex I/Fab co-complex crystals represent a space group different to the one observed for the native protein. Ongoing experiments are aimed at further enhancements of the diffraction quality of the crystals. Providing a different space group the CI/Fab co-complexes may become a very useful approach for structure determination of the enzyme. Moreover, the bound Fab offers an additional possibility to generate phase information. The antibody-mediated crystallization represents a valuable tool in structural characterization of the NADH:oxidoreductase subcomplexes or even single subunits. 2. UDP-glucose pyrophosphorylase UDP-glucose pyrophosphorylase from Yarrowia lipolytica displays affinity towards Ni2+ NTA and was first detected in a contaminated sample of complex I. Following, separation from complex I, Ugp1p was purified using anion exchange chromatography. Sequence similarity studies revealed high identity to other known pyrophosphorylases. As indicated by laser-based mass spectrometry method (LILBID) Ugp1p from Y. lipolytica builds octamers similarly to the enzyme from Saccharomyces cerevisiae. The initial crystals grew as thin needles favorably in sitting drop setups. The size of the crystals was increased by employment of a micro batch technique. The improved crystals diffracted X-rays to a resolution of 3.2 Å at the synchrotron beamline. Structural characterization is under way using a molecular replacement approach based on the published structure of baker’s yeast UGPase.