Ionen—VIII : Das tetraphenyloborat-ion als kernresonanzverschiebungsreagens bei quartären ammonium-kationen

B(C₆H₅)₄^? is a useful NMR shift reagent for quaternary ammonium cations. The signals of N^?CH protons are shifted to higher field by up to 4·2 ppm. More distant protons experience a much smaller shielding. Within certain limits, the signal position can be chosen arbitrarily. The utility of salt mixtures for signal assignment is demonstrated.     

1H NMR detection of immobilized water molecules within a strong distal hydrogen-bonding network of substrate-bound human heme oxygenase-1

Solution 1H NMR is used to probe the environments of the donor protons of eight strong hydrogen bonds on the distal side of the heme substrate in the cyanide-inhibited, substrate-bound complex of human heme oxygenase, hHO. It is demonstrated that significant magnetization transfer from the bulk water signal to the eight labile protons does not result from chemical exchange, but from direct nuclear Overhauser effect due to the dipolar interaction of these labile protons with "ordered" water molecules. The enzyme labile proton to water proton distances are estimated at approximately 3 A. It is proposed that the role of the strong hydrogen-bonding network is to immobilize numerous water molecules which both stabilize the activated hydroperoxy species and funnel protons to the active site.     

Changes in the heme ligation during folding of a Geobacter sulfurreducens sensor GSU0935

Ligand binding and substitution reactions are important for metalloprotein folding and function. The heme sensor of a methyl-accepting chemotaxis GSU0935 is a c-type cytochrome from the bacterium Geobacter sulfurreducens. The heme domain switches one of its axial ligands from H(2)O to a low-spin ligand, presumably Met, upon reduction. The study analyzes the stability and folding kinetics of the ferric domain. Guanidine hydrochloride denaturation yields the low-spin heme species arising from coordination of the ferric heme by non-native His residues. The population of the low-spin species further increases and then declines during protein refolding. Kinetics and mutational effects suggest that His54, from the N-terminal region of the domain, is the transient ligand to the heme. The capture and release of a non-native ligand within the compact partially-folded structures illustrates the flexibility of the heme environment in GSU0935, which may relate to the domain sensor function.     

1,3‐dipolar cycloaddition reactions of 2‐substituted azomethine N‐oxides with N‐benzyl maleimides leading to the synthesis of stereoisomers

The azomethine N‐oxides ( 1 ) on reacting with N‐benzylmaleimide ( 2 ) provide a mixture of stereoisomers 2,3‐diphenyl‐5‐benzyl‐4H‐2,3,3a,5,6,6a‐hexahydropyrrolo[3,4‐d]isoxazole‐4,6–dione derivatives ( 3 ) in good yields. These isomers have been assigned cis and trans configurations ( 3‐A and 3‐B ) with respect to proton C₃‐H on the azomethinic carbon on the basis of their PMR and H‐NMR COSY data. The ratio between cis and trans isomers has been found to be dependent on substituents present at ortho position of C‐phenyl aldehydic moiety. The salient feature of these 1,3‐dipolar cycloaddition reactions lies in that the benzylic protons on N‐benzyl moiety suffer gem coupling, indicating magnetic nonequivalence. J. Heterocyclic Chem.,, (2012).     

Measurement of Very Fast Exchange Rates of Individual Amide Protons in Proteins by NMR Spectroscopy

NMR spectroscopy is a pivotal technique to measure hydrogen exchange rates in proteins. However, currently available NMR methods to measure backbone exchange are limited to rates of up to a few per second. To raise this limit, we have developed an approach that is capable of measuring proton exchange rates up to approximately 10⁴ s⁻¹. Our method relies on the detection of signal loss due to the decorrelation of antiphase operators 2N_xH_z by exchange events that occur during a series of pi pulses on the ¹⁵N channel. In practice, signal attenuation was monitored in a series of 2D H(CACO)N spectra, recorded with varying pi-pulse spacing, and the exchange rate was obtained by numerical fitting to the evolution of the density matrix. The method was applied to the small calcium-binding protein Calbindin D_9k, where exchange rates up to 600 s⁻¹ were measured for amides, where no signal was detectable in ¹⁵N−¹H HSQC spectra. A temperature variation study allowed us to determine apparent activation energies in the range 47–69 kJ mol⁻¹ for these fast exchanging amide protons, consistent with hydroxide-catalyzed exchange.     

Folding behaviors of apocytochrome b5 and its mutants: Insights from high temperature molecular dynamics simulations

Apocytochrome b₅ (apocyt b₅) with heme removal from the heme-binding core 1, and its mutants with amino acid replaced in the hydrophobic core 2, namely apocyt b₅ Y7P, P81A and H15R/S20E, have been subjected to molecular dynamics (MD) simulation at high temperature (500 K) for elucidating their folding behaviors. The early events upon thermal induced unfolding were found to be in good agreement with available experimental results, and the lowest stability of Y7P was predicted among the four apoproteins. The influences of these key residues on protein folding behavior were compared directly at an atomic level. At the same time, the influences of non-native interactions of hydrogen bonds and salt-bridges on protein stabilities were analyzed in detail. The insights from current MD simulations are valuable for understanding the apoprotein folding and the holoprotein formation in terms of heme proteins.     

Hydrogen-bonding partner of the proton-conducting histidine in the influenza m2 proton channel revealed from (1)h chemical shifts

The influenza M2 protein conducts protons through a critical histidine (His) residue, His37. Whether His37 only interacts with water to relay protons into the virion or whether a low-barrier hydrogen bond (LBHB) also exists between the histidines to stabilize charges before proton conduction is actively debated. To address this question, we have measured the imidazole (1)H(N) chemical shifts of His37 at different temperatures and pH using 2D (15)N-(1)H correlation solid-state NMR. At low temperature, the H(N) chemical shifts are 8-15 ppm at all pH values, indicating that the His37 side chain forms conventional hydrogen bonds (H-bonds) instead of LBHBs. At ambient temperature, the dynamically averaged H(N) chemical shifts are 4.8 ppm, indicating that the H-bonding partner of the imidazole is water instead of another histidine in the tetrameric channel. These data show that His37 forms H-bonds only to water, with regular strength, thus supporting the His-water proton exchange model and ruling out the low-barrier H-bonded dimer model.     

Quality check of heparin injections by 1H-nuclear magnetic resonance spectroscopy

The quality of commercial heparin injections was examined by 400-MHz proton nuclear magnetic resonance (1H-NMR) spectroscopy using several measuring modes. The signals of the N-acetyl protons, as well as the sugar-ring protons, attached to the sulfamino and sulfato group-bearing carbons could be easily distinguished from other proton signals and quantified. Measuring at a high temperature (60 degrees C) enabled clear isolation of the H-5 proton signal in the sulphated iduronic acid residue (Is-5) from other proton signals including that of water. The heparin contents of various heparin injections were estimated by using this signal as an index. However, the signal intensity was not parallel with anticoagulant activity. On the other hand, the N-acetyl proton signal was highly correlated to anticoagulant activity. The present method was also useful for concurrent identification of additives in heparin injections.     

Ionen-VI: Das Tetraphenyloborat-Ion als Kernresonanz-Verschiebungsreagens bei Anilinium-Kationen

B(C₆H₅)₄? is a useful NMR shift reagent for anilinium cations. The signals of N⊕? C? H protons are shifted to higher field by 1.1 to 2.5 ppm. More distant protons experience a much smaller shielding. Within certain limits the signal position can be chosen arbitrarily. The utility of salt mixtures for the resolution of accidental signal coincidences and signal assignment is demonstrated.     

Paramagnetic 1H NMR spectrum of nickel(II) pseudoazurin: investigation of the active site structure and the acid and alkaline transitions

The paramagnetic (1)H NMR spectrum of Ni(II) pseudoazurin [(PA)Ni(II)] possesses a number of resonances exhibiting sizable Fermi-contact shifts. These have been assigned to protons associated with the four ligating amino acids, His40, Cys78, His81, and Met86. The shifts experienced by the C(gamma)H protons of the axial Met86 ligand are unprecedented compared to other Ni(II)- and Co(II)-substituted cupredoxins (the C(gamma)(1)H signal is found at 432.5 ppm at 25 degrees C). The large shift of protons of the axial Met86 ligand highlights a strong Ni(II)-S(Met) interaction in (PA)Ni(II). The paramagnetic (1)H NMR spectrum of (PA)Ni(II) is altered by decreasing and increasing the pH value from 8.0. At acidic pH a number of the hyperfine-shifted resonances undergo limited changes in their chemical shift values. This effect is assigned to the surface His6 residue whose protonation results in a structural modification of the active site. Increasing the pH value from 8.0 has a more significant effect on the paramagnetic (1)H NMR spectrum of (PA)Ni(II), and the alkaline transition can now be assigned to two surface lysine residues close to the active site of the protein. The effect of altering pH on the (1)H NMR spectrum of Ni(II) pseudoazurin is smaller than that previously observed in the Cu(II) protein indicating more limited structural rearrangements at the non-native metal site.     

1H NMR studies on intramolecular hydrogen bonding in perchlorates of N-(pyridyl)amides of 6-methylpicolinic acid N-oxide

The ¹H NMR spectra of perchlorates of N-(pyridyl)amides of 6-methylpicolinic acid N-oxide (PYAP) in CD₃CN at 100 MHz show two proton signals belonging to two distinct intramolecular hydrogen bonds. The position of these signals is independent of concentration and temperature. That of the proton of the N? H ?O bond in PYAP is shifted to still lower field than in N-(pyridyl)amides of 6-methylpicolinic acid N-oxide (PYA) due to the inductive effect of the pyridine cation and the formation of another intramolecular hydrogen N⁺? H ?O bond. The proton of the N⁺? H ?O bond interacts strongly with its environment and is highly sensitive to traces of water. Presumably, water leads to dissociation of the intramolecular bond.     

A DFT study of the mechanism of Ni superoxide dismutase (NiSOD): role of the active site cysteine-6 residue in the oxidative half-reaction

In the present DFT study, the catalytic mechanism of H2O2 formation in the oxidative half-reaction of NiSOD, E-Ni(II) + O2- + 2H+ --> E-Ni(III) + H2O2, has been investigated. The main objective of this study is to investigate the source of two protons required in this half-reaction. The proposed mechanism consists of two steps: superoxide coordination and H2O2 formation. The effect of protonation of Cys6 and the proton donating roles of side chains (S) and backbones (B) of His1, Asp3, Cys6, and Tyr9 residues in these two steps have been studied in detail. For protonated Cys6, superoxide binding generates a Ni(III)-O2H species in a process that is exothermic by 17.4 kcal/mol (in protein environment using the continuum model). From the Ni(III)-O2H species, H2O2 formation occurs through a proton donation by His1 via Tyr9, which relative to the resting position of the enzyme is exothermic by 4.9 kcal/mol. In this pathway, a proton donating role of His1 residue is proposed. However, for unprotonated Cys6, a Ni(II)-O2- species is generated in a process that is exothermic by 11.3 kcal/mol. From the Ni(II)-O2- species, the only feasible pathway for H2O2 formation is through donation of protons by the Tyr9(S)-Asp3(S) pair. The results discussed in this study elucidate the role of the active site residues in the catalytic cycle and provide intricate details of the complex functioning of this enzyme.     

A novel hydrogen-bonding N-oxide–sulfonamide–nitro N—H…O synthon determining the architecture of benzenesulfonamide cocrystals

The structures of novel cocrystals of 4-nitropyridine N-oxide with benzenesulfonamide derivatives, namely, 4-nitrobenzenesulfonamide–4-nitropyridine N-oxide (1/1), C₅H₄N₂O₃·C₆H₆N₂O₄S, and 4-chlorobenzenesulfonamide–4-nitropyridine N-oxide (1/1), C₆H₆ClNO₂S·C₅H₄N₂O₃, are stabilized by N—H…O hydrogen bonds, with the sulfonamide group acting as a proton donor. The O atoms of the N-oxide and nitro groups are acceptors in these interactions. The latter is a double acceptor of bifurcated hydrogen bonds. Previous studies on similar crystal structures indicated competition between these functional groups in the formation of hydrogen bonds, with the priority being for the N-oxide group. In contrast, the present X-ray studies indicate the existence of a hydrogen-bonding synthon including N—H…O(N-oxide) and N—H…O(nitro) bridges. We present here a more detailed analysis of the N-oxide–sulfonamide–nitro N—H…O ternary complex with quantum theory computations and the Quantum Theory of Atoms in Molecules (QTAIM) approach. Both interactions are present in the crystals, but the O atom of the N-oxide group is found to be a more effective proton acceptor in hydrogen bonds, with an interaction energy about twice that of the nitro-group O atoms.     

Oxygen‐binding Protein Fiber and Microgel: Supramolecular Myoglobin–Poly(acrylate) Conjugates

A supramolecular conjugate of myoglobin (Mb) and water‐soluble poly(acrylate), (PA_5k and PA_25k, where 5k and 25k represent the molecular weight of the polymers, respectively), is constructed on the basis of a noncovalent heme‐heme pocket interaction. The modified heme with an amino group linked to the terminus of one of the heme‐propionates is coupled to the side‐chain carboxyl groups of poly(acrylate) activated by N‐hydroxysuccinimide. The ratios of the heme‐modified monomer unit and the unmodified monomer unit (m:n) in the polymer chains of Heme‐PA_5k and Heme‐PA_25k were determined to be 4.5:95.5 and 3.1:96.9, respectively. Subsequent addition of apoMb to the conjugated polymers provides Mb‐connected fibrous nanostructures confirmed by atomic force microscopy. A mixture of the heme‐modified polymer and dimeric apomyoglobin as a cross‐linker forms a microgel in which the reconstituted myoglobin retains its native exogenous ligand binding activity.     

Kinetics of histidine deligation from the heme in GuHCl-unfolded Fe(III) cytochrome C studied by a laser-induced pH-jump technique.

We have developed an instrumental setup that uses transient absorption to monitor protein folding/unfolding processes following a laser-induced, ultrafast release of protons from o-nitrobenzaldehyde. The resulting increase in [H(+)], which can be more than 100 microM, is complete within a few nanoseconds. The increase in [H(+)] lowers the pH of the solution from neutrality to approximately 4 at the highest laser pulse energy used. Protein structural rearrangements can be followed by transient absorption, with kinetic monitoring over a broad time range (approximately 10 ns to 500 ms). Using this pH-jump/transient absorption technique, we have examined the dissociation kinetics of non-native axial heme ligands (either histidine His26 or His33) in GuHCl-unfolded Fe(III) cytochrome c (cyt c). Deligation of the non-native ligands following the acidic pH-jump occurs as a biexponential process with different pre-exponential factors. The pre-exponential factors markedly depend on the extent of the pH-jump, as expected from differences in the pK(a) values of His26 and His33. The two lifetimes were found to depend on temperature but were not functions of either the magnitude of the pH-jump or the pre-pulse pH of the solution. The activation energies of the deligation processes support the suggestion that GuHCl-unfolded cyt c structures with non-native histidine axial ligands represent kinetic traps in unfolding.     

Study of the structure and stereochemistry of flavonoid O-rhamnosides with the aid of PMR spectroscopy

The PMR method has been used in the study of the structure and stereochemistry of a number of flavonoid rhamnosides. The systematic study of the PMR spectra of the native compounds and their TMS ethers and complete acetates has permitted the size of the oxide ring, the conformation of the rhamnose residue, and, in combination with the results of polarimetric analysis, the configuration of the glycosidic bond to be determined. Information on the size of the oxide ring is given by — in addition to, the J_{2, 3} proton coupling constant — the signals of the protons at C-5 in the spectra of the full acetates. In the diagnostic region of 3.2–4.0 ppm, only the proton H-5 resonates in the rhamnopyranosides, while for the rhamnofuranosides a different structure of the H-4 signal must be expected. It has been shown that all the compounds studied (more than 20 specimens) are α-L-rhamnopyranosides and have the¹C₄ conformation of the sugar residue. In view of this, the hypothesis has been expressed that the existence of natural flavonoid rhamnosides in the furanoside form is improbable.     

470—The proton pump at work: Part I. The basic concept of excess proton/defect proton translocation

The Nagle-Morowitz proton pump, which is based on proton transport in water and ice, is shown to be inapplicable to weakly H-bonded proton transport systems. It is demonstrated that in weakly or non H-bonded systems protons can migrate either as excess protons H using a high-lying proton conduction band or as defect protons H′ using intermediate energy levels. As H? the protons act as positive charge carriers endowed with a very high mobility. As H′ they act as negative charge carriers. The H′ transport mechanism involves thermally activated proton tunneling. Owing to the very large mobility differences between H? and H′, > 10⁶, very high potentials can be self-generated in any situation which creates a concentration gradient. Proton conductivity data on inorganic model compounds are presented. Applying these results to proton transport across biomembranes, transmembrane potentials U_m, acidification Δ pH and transient phenomena can be explained as result of H? and H′ translocation.     

1H NMR studies of intramolecular hydrogen bonding in N-(pyridyl)amides of 6-methylpicolinic acid N-oxide

The ¹H NMR spectra of seven N-(pyridyl)amides of 6-methylpicolinic acid N-oxide in chloroform were obtained. The influence on the chemical shifts of the N? H protons of temperature, concentration and the CH₃ substituent in the pyridine ring was studied. The N? H protons were found to be shifted to low fields (～14 ppm) owing to the formation of strong intramolecular hydrogen bonding. The influence of the pyridine ring on the chemical shift of the N? H proton is comparable with the inductive effect of the p-nitrophenyl group. The hindered rotation around the N-pyridyl bond of N-(α-pyridyl)amides of 6-methylpicolinic acid in solution is discussed.     

细胞色素b5突变体V45H NMR初步研究

通过分析在H2O和D2O中采集,DQF-COSY,TOCSY和NOESY等二维核磁共振波谱鉴定了细胞色素b5定点突变体V45H(残基Val^45突变为His^45)的大多数氨基酸残基的质子自旋系统,通过解析NOESY谱中的dNN(i,i+1),dαN(i,i+1),dαN(i,i+2),dαN(i,i+3),dαβ(i,i+3)和dβN(i,i+1)等NOE相关,完成了其序列特异性归属以及主链和侧链质子共振信号的全归属。突变体V45H的二级结构分析表明残基Val^45突变为His^45对分子的整体折叠影响不大。但是,与野生型细胞色素b5相比较,突变体V45H主链酰胺质子的化学位移指数提示突变使其血红素疏水腔的微环境受到扰动。以上实验结果为进一步测定V45H的溶液结构和分析残基Val^45在蛋白质中的作用提供了基础。