Remote Water-Mediated Proton Transfer Triggers Inter-Cu Electron Transfer: Nitrite Reduction Activation in Copper-Containing Nitrite Reductase

The copper-containing nitrite reductase (CuNiR) catalyzes the biological conversion of nitrite to nitric oxide; key long-range electron/proton transfers are involved in the catalysis. However, the details of the electron-/proton-transfer mechanism are still unknown. In particular, the driving force of the electron transfer from the type-1 copper (T1Cu) site to the type-2 copper (T2Cu) site is ambiguous. Here, we explored the two possible proton-transfer channels, the high-pH proton channel and the primary proton channel, by using two-layered ONIOM calculations. Our calculation results reveal that the driving force for electron transfer from T1Cu to T2Cu comes from a remote water-mediated triple-proton-coupled electron-transfer mechanism. In the high-pH proton channel, the water-mediated triple-proton transfer occurs from Glu113 to an intermediate water molecule, whereas in the primary channel, the transfer is from Lys128 to His260. Subsequently, the two channels employ another two or three distinct proton-transfer steps to deliver the proton to the nitrite substrate at the T2Cu site. These findings explain the detailed proton-/electron-transfer mechanisms of copper-containing nitrite reductase and could extend our understanding of the diverse proton-coupled electron-transfer mechanisms in complicated proteins.     

Direct electrochemistry of dinuclear CuA fragment from cytochrome c oxidase of Thermus thermophilus at surfactant modified glassy carbon electrode

Cytochrome c oxidase is ubiquitous enzyme involved in the terminal step of respiratory electron transfer process. The unique binuclear copper center containing bis-dithiolato bridges form a valance delocalized [Cu^1.5+-Cu^1.5+] state of the metal center located at the subunit II of cytochrome c oxidase. This metal center acts as the electron entry site of the enzyme and accepts electrons from cytochrome c. Direct electrochemistry of this binuclear copper center containing the water soluble protein obtained by genetically truncating the membrane bound part of the subunit II from Thermus thermophilus was achieved by favorable orientation of the protein on glassy carbon electrode surface promoting efficient electron transfer in the presence of various surfactants. Very reproducible, Nernstian responses are obtained with Cu_A. The redox potential and the electrochemical response were enhanced prominently in case of cationic surfactant CTAB indicating that the nature of the surfactant has a significant effect on the microenvironment of the protein-electrode interface. The results have been used to understand the mechanism of electron transfer from cytochrome c to the copper center during the enzymatic reaction.     

Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks

Respiratory oxidases are transmembrane enzymes that catalyze the reduction of dioxygen to water in the final step of aerobic respiration. This process is linked to proton pumping across the membrane. Here, we developed a method to study the catalytic turnover of the quinol oxidase, cytochrome bo₃ from E. coli at single-molecule level. Liposomes with reconstituted cytochrome bo₃ were loaded with a pH-sensitive dye and changes in the dye fluorescence, associated with proton transfer and pumping, were monitored as a function of time. The single-molecule approach allowed us to determine the orientation of cytochrome bo₃ in the membrane; in ∼70 % of the protein-containing liposomes protons were released to the outside. Upon addition of substrate we observed the buildup of a ΔpH (in the presence of the K⁺ ionophore valinomycin), which was stable over at least ∼800 s. No rapid changes in ΔpH (proton leaks) were observed during steady state proton pumping, which indicates that the free energy stored in the electrochemical gradient in E. coli is not dissipated or regulated through stochastic transmembrane proton leaks, as suggested from an earlier study (Li et al. J. Am. Chem. Soc. (2015) 137, 16055–16063).     

Computational Study on the Catalytic Reaction Mechanism of Heme Haloperoxidase Enzymes

Heme haloperoxidases are unique enzymes in biology that react H₂O₂ and halides on a heme center to generate hypohalide, which reacts with a substrate by halide transfer. We studied model complexes of the active site of heme haloperoxidase and investigated the reaction mechanism starting from an iron(III)-hydrogen peroxide-heme complex. We find two stepwise proton transfers by active site Glu and His residues to form Compound I and water, whereby the second proton transfer step is rate-determining. In a subsequent reaction with chloride the oxygen atom transfer is studied to form hypohalide. Overall, the free energy of activation of the second proton transfer and oxygen atom transfer to halide are similar in energy with free energies of activation of around 20 kcal mol⁻¹. The calculations show that during oxygen atom transfer from Compound I to halide, significant charge-transfer happens prior to the transition state. This implies that the reaction may be enhanced in polar environments and through second-coordination sphere effects. The studies show that the conversion of H₂O₂ and halide on a heme center is fast and few intermediates along the reaction mechanism will have a lifetime that is long enough to enable trapping and characterization with experimental methods. A range of active site models and density functional theory methods were tested, but little effect is seen on the mechanism and optimized geometries.     

Does Hydride Ion Transfer from Silanes to Carbenium Ions Proceed Via a Rate-Determining Formation of a Silicenium Ion or Via a Rate-Determining Electron Transfer? An Ab Initio Quantum Mechanical Study and a Curve-Crossing Analysis

The hydride transfer reactions from simple silanes to carbenium ions are studied by ab initio calculations. The simplest reaction, H₄Si + CH₃⁺ → H₃Si⁺ + CH₄, is also studied with inclusion of the solvent effect (with the SCRF method) in the ab initio scheme. Under all conditions the preferred mechanism is the synchronous hydride transfer (SHT), which is barrierless in the gas phase but possesses small barriers in solution. The mechanistic alternative involving a rate-determining single electron transfer (SET) step followed by H-atom abstraction is found to be of very high energy. Modelling of the primary isotope effect for the SHT process of H₃SiH(D) + CH₃* → H₃Si⁺ + H₃CH(D) shows that the primary isotope effect is small, between ca. 1.1 and 2.7, for the entire relevant range of Si—H(D) distances (1.5–2.3 Å). Furthermore, the pattern of the computed primary isotope effect shows it to be an insensitive probe of the SHT mechanism. The curve-crossing method is used to model the mechanistic dichotomy. It is shown that the reaction profiles for both SHT and SET arise from an avoided crossing between the ground state and a charge transfer state of the R₃SiH//R′₃C⁺ reactant pair. Thus, in the SHT mechanism a single electron switches sites in synchronicity with bond reorganization, while in SET the electron switch precedes the bond coupling. This avoided bond coupling is the foremost disadvantage of the SET mechanism. The common origin of the avoided crossing elucidates the reason why SHT exhibits characteristics of an electron transfer process without actually being a SET process.     

Involvement of Acidic Amino Acid Residues in Zn2+ Binding to Respiratory Complex I

Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn²⁺ ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn²⁺inhibition of the proton‐pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm^?1) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn²⁺ binding. In variant D563N^L, which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn²⁺ binding is changed. Our data support a role for this residue in proton translocation.     

Direct voltammetry and catalysis of hemoenzymes in methyl cellulose film

Horseradish peroxidase (HRP) and catalase (Cat) immobilized on edge-plane pyrolytic graphite (EPG) electrodes by methyl cellulose (MC). Both the hemoenzymes entrapped in the MC film undergo fast direct electron-transfer reaction, corresponding to hemeFe(III)+e→hemeFe(II). The formal potential (E⁰′), the apparent coverage (Γ), the electron transfer coefficient (α) and the apparent electron transfer rate constant (k_s) were calculated by performing nonlinear regression analysis of square wave voltammetry (SWV) experimental data. E⁰′ are linearly dependent on pH, indicating the electron transfer of Fe(III)/Fe(II) redox couple companied with the transfer of proton. The processes of catalytically reducing oxygen, hydrogen peroxide and nitric oxide by HRP and Cat entrapped in MC film are also explored.     

Electron Donation in Photosystem II

The pathways of electron donation in Photosystem II (PS II) as studied by electron paramagnetic resonance (EPR) and time-resolved optical spectroscopy are discussed. The EPR studies have defined two competing pathways of electron donation in PS II, from cytochrome b₅₅₉ and from the Mn site of the oxygen-evolving center. The kinetics of re-reduction of the primary electron donor of PS II (P₆₈₀). as measured by optical spectroscopy, are re-evaluated in light of the EPR results. We propose that the 35-μs kinetic component is due to the reduction of chlorophyll, an alternate electron donor on the cytochrome b₅₅₉ pathway, rather than to the reduction of P₆₈₀. The chlorophyll/cytochrome b₅₅₉ pathway has been proposed to be part of a cyclic electron transfer pathway around PS II; we suggest that photooxidation of chlorophyll is the first step leading to photoinhibition and that cytochrome b₅₅₉ serves to protect PS II from photoinhibition by rapidly re-reducing the oxidized chlorophyll (Thompson, L.K.; Brudvig, G.W. Biochemistry, 1988, 27 : 6653). These results and proposals are summarized in an overall scheme of electron transfer pathways and rates in PS II.     

Hydrogen Bond Compression during Triple Proton Transfer in Crystalline Pyrazoles. A Dynamic 15N NMR Study

Using dynamic solid state ¹⁵N CPMAS NMR spectroscopy (CP ≡ cross polarization, MAS ≡ magic-angle spinning), the kinetics of degenerate intermolecular triple proton and deuteron transfers in the cyclic trimers of ¹⁵N-labeled polycrystalline 4-nitropyrazole (4NO₂P) and 4-bromopyrazole (4BrP) have been studied as a function of temperature and are compared to the kinetics of triple proton transfer in bulk solid 3,5-dimethylpyrazole (DMP) studied previously. The results show that the transfer kinetics in the new trimers are much faster than in DMP. However, the kinetic HHH/HHD/HDD/DDD isotope effects of 4NO₂P are similar to those of DMP. These effects indicate a single barrier for the triple proton transfers where all three protons lose zero-point energy in the transition state, as expected for a structure with three compressed hydrogen bonds. At low temperatures, strong deviations from an Arrhenius-behavior are observed which are described in terms of a modified Bell tunneling model and a concerted proton motion. The barrier for the triple proton transfer in 4NO₂P and 4BrP is substantially smaller than in DMP. As there is no correlation with the electronic properties of the substituents, we assign this finding to steric effects where the bulky methyl groups of DMP in the 3- and 5-positions hinder the hydrogen bond compression, in contrast to 4NO₂P and 4BrP exhibiting substitutents in the 4-position. These results lead to a minimum energy pathway of the proton transfer following in the absence of steric hindering the hydrogen bond correlation line q₁ = f(q₂), established previously, where q₁ represents the deviation of the proton from the hydrogen bond center and q₂ the N…N distance. Tunneling occurs at constant N…N distances.     

Construction of continuous proton-conduction channels through polyvinylimidazole nanotubes to enhance proton conductivity of polymer electrolyte membrane

Proton-exchange membranes (PEMs) with high proton conductivity and low cost are crucial for the commercial promotion of proton-exchange membrane fuel cells. In this study, inspired by the mechanism of plant ducts transporting moisture and biological proton transfer, polyvinylimidazole nanotubes (PVINTs) are prepared by a simple template method and then incorporated into a sulfonated poly(aryl ether sulfone) (SPES) matrix to fabricate composite membranes (SPES/PVINTs-X, where X is PVINT content in percent). The membranes were fully characterized using field emission scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and mechanical testing. The results indicated that the incorporation of PVINTs could improve the mechanical performance and dimensional stability of the membranes. In particular, the SPES/PVINTs-7.5 composite membrane achieves remarkable results of proton conductivity of 0.212 S/cm under fully hydrated conditions at 80 °C, which is 56% higher than that of the SPES membrane. The construction of proton-transfer channels through polymer nanotubes described in this paper may provide new insights into the preparation of composite proton-exchange membranes. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47106.     

Recent Advances in Structural Studies of Cytochrome bd and Its Potential Application as a Drug Target

Cytochrome bd is a triheme copper-free terminal oxidase in membrane respiratory chains of prokaryotes. This unique molecular machine couples electron transfer from quinol to O₂ with the generation of a proton motive force without proton pumping. Apart from energy conservation, the bd enzyme plays an additional key role in the microbial cell, being involved in the response to different environmental stressors. Cytochrome bd promotes virulence in a number of pathogenic species that makes it a suitable molecular drug target candidate. This review focuses on recent advances in understanding the structure of cytochrome bd and the development of its selective inhibitors.     

The Reduction of Dioxygen by Keggin Heteropolytungstates

Reversible redox chemistries are an inherent feature of numerous metal oxide cluster anions (POMs). Moreover, as discrete molecular structures with well-defined and controllable solution chemistries, POMs can be deployed as physicochemical probes for studying inorganic reaction mechanisms. In the past decade, we have used an iso-structural series of α-Keggin heteropolytungstate cluster anions to systematically investigate a number of fundamental topics, including electron transfer to dioxygen. The iso-structural series of cluster anions is obtained by varying the heteroatom, Xⁿ⁺, in the plenary, T_d-symmetry α-Keggin ion, Xⁿ⁺W₁₂O₄₀⁽⁸⁻ⁿ⁾⁻, from Al³⁺ to Si⁴⁺ to P⁵⁺. This results in a stepwise and linear modulation of ion charge and reduction potential, whose concerted effects on reaction rates can be used to better understand electron-transfer processes. Starting from the acquisition of activation parameters associated with electron self-exchange between the POMs themselves, the studies discussed in this review provide a detailed account of electron transfer from reduced α-Keggin heteropolytungstate anions to dioxygen, culminating in the recent discovery of a fundamentally new mechanism for electron transfer to O₂ in water.     

Exploring Electron/Proton Transfer and Conformational Changes in the Nitrogenase MoFe Protein and FeMo-cofactor Through Cryoreduction/EPR Measurements

We combine cryoreduction/annealing/EPR measurements of nitrogenase MoFe protein with results of earlier investigations to provide a detailed view of the electron/proton transfer events and conformational changes that occur during early stages of [e⁻/H⁺] accumulation by the MoFe protein. This includes reduction of: 1) the non-catalytic state of the iron-molybdenum cofactor (FeMo-co) active site that is generated by chemical oxidation of the resting-state cofactor (S=3/2) within resting MoFe (E₀); and 2) the catalytic state that has accumulated n=1 [e⁻/H⁺] above the resting-state level, denoted E₁(1H) (S≥1) in the Lowe-Thorneley kinetic scheme. FeMo-co does not undergo a major change of conformation during reduction of oxidized FeMo-co. In contrast, FeMo-co undergoes substantial conformational changes during the reduction of E₀ to E₁(1H), and of E₁(1H) to E₂(2H) (S=3/2). The experimental results further suggest that the E₁(1H)→E₂(2H) step involves coupled delivery of a proton and an electron (PCET) to FeMo-co of E₁(H) to generate a nonequilibrium S= form E₂(2H)*. This subsequently undergoes conformational relaxation and attendant change in the FeMo-co spin state, to generate the equilibrium E₂(2H) (S=3/2) state. Unexpectedly, these experiments also reveal conformational coupling between FeMo-co and the P cluster, and between the Fe protein binding and FeMo-co, which might play a role in gated electron transfer from reduced Fe protein to FeMo-co.     

STD‐NMR Studies Suggest that Two Acceptor Substrates for GlfT2, a Bifunctional Galactofuranosyltransferase Required for the Biosynthesis of Mycobacterium tuberculosis Arabinogalactan,Compete for the Same Binding Site

The mycobacterial cell wall is a complex architecture, which has, as its major structural component, a lipidated polysaccharide covalently bound to peptidoglycan. This structure, termed the mycolyl–arabinogalactan–peptidoglycan complex, possesses a core galactan moiety composed of approximately 30 galactofuranosyl (Galf) resides attached via alternating β‐(1→6) and β‐(1→5) linkages. Recent studies have shown that the entire galactan is synthesized by the action of only two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We report here saturation‐transfer difference (STD) NMR spectroscopy studies with GlfT2 using two trisaccharide acceptor substrates, β‐D ‐Galf‐(1→6)‐β‐D ‐Galf‐(1→5)‐β‐D ‐Galf‐O(CH₂)₇CH₃ ( 2 ) and β‐D ‐Galf‐(1→5)‐β‐D ‐Galf‐(1→6)‐β‐D ‐Galf‐O(CH₂)₇CH₃ ( 3 ), as well as the donor substrate for the enzyme, UDP‐Galf. Competition STD‐NMR titration experiments and saturation transfer double difference (STDD) experiments with 2 and 3 were undertaken to explore the bifunctionality of this enzyme, in particular to answer whether one or two active sites are responsible for the formation of both β‐(1→5)‐ and β‐(1→6)‐Galf linkages. It was demonstrated that 2 and 3 bind competitively at the same site; this suggests that GlfT2 has one active site pocket capable of catalyzing both β‐(1→5) and β‐(1→6) galactofuranosyl transfer reactions. The addition of UDP‐Galf to GlfT2 in the presence of either 2 or 3 generated a tetrasaccharide product; this indicates that the enzyme was catalytically active under the conditions at which the STD‐NMR experiments were carried out.     

Cover Feature: Superior Proton-Transfer Catalytic Promiscuity of Cytochrome c in Self-Organized Media (7/2021)

Evolutionarily elderly proteins commonly feature greater catalytic promiscuity. Cytochrome c is among the first set of proteins in evolution to have known prospects in electron transport and peroxidative properties. Here, we report that cyt c is also a proficient proton-transfer catalyst and enhances the Kemp elimination (KE; model reaction to show proton transfer catalytic property) by ∼750-fold on self-organized systems like micelles and vesicles. The self-organized systems mimic the mitochondrial environment in vitro for cyt c. Using an array of biophysical and biochemical mutational assays, both acid–base and redox mechanistic pathways have been explored. The histidine moiety close to hemin group (His18) is mainly responsible for proton abstraction to promote the concerted E2 pathway for KE catalysis when cyt c is in its oxidized form; this has also been confirmed by a H18A mutant of cyt c. However, the redox pathway is predominant under reducing conditions in the presence of dithiothreitol over the pH range 6–7.4. Interestingly, we found almost 750-fold enhanced KE catalysis by cyt c compared to aqueous buffer. Overall, in addition to providing mechanistic insights, the data reveal an unprecedented catalytic property of cyt c that could be of high importance in an evolutionary perspective considering its role in delineating the phylogenic tree and also towards generating programmable designer biocatalysts.     

2D‐ and 3D‐Photoluminescence Imaging of Eu(III) Embedded in CeO2 Nanomatrix

2D‐ and 3D‐photoluminescence characteristics of Eu(III) doped in CeO₂ nanoparticles were fully imaged for the first time. Their fundamental natures were also examined by scanning electron microscopy (SEM), X‐ray diffraction (XRD) crystallography, and UV–visible absorption spectroscopy. The magnetic dipole ⁵D₀ → 7F₁ transition was dominated by an indirect transition associated with a O^2?–Ce⁴⁺ charge‐transfer band of CeO₂. The electric dipole ⁵D₀ → ⁷F₂ transition was dominated by a direct transition of Eu(III), indicating that Eu(III) replaces Ce(IV) at octahedral sites (O_h and O) with and without an inversion center. Upon annealing, the photoluminescence intensity caused by direct transition was dramatically decreased, whereas that induced by indirect transition was greatly enhanced. These findings indicate that charge transfer to the Eu(III) at the octahedral (O_h) site with the inversion center is more efficient than that to the Eu(III) site without an inversion center. The absolute quantum yield for the 10 mol% Eu(III)–CeO₂ was found to be ? = 0.007 at an excitation wavelength of 350 nm. The photoluminescence of Tb‐doped CeO₂ was briefly discussed for comparison.     

Different Hemi-Salen/Salan Ligand Containing Binuclear Boron-Fluoride Complexes: Synthesis,Spectroscopy, Fluorescence Properties,and Catalysis

A family of hemi-salen (L₁H–L₆H) and hemi-salan (L_1aH–L_2aH) ligands-based N,O-chelated binuclear boron-fluoride [L_n(BF₂)₂] (n = L₁–L₆ or L_1a–L_2a) complexes have been prepared and characterized by a variety of spectroscopic techniques (¹H, ¹³C and ¹⁹F NMR, FT-IR, UV-Vis, LC-MS, and fluorescence spectra) and elemental analysis. All of the binuclear boron-fluoride complexes exhibit strong absorption bands due to S₀→S₁ transitions and strong fluorescence properties were observed at room temperature in the solution. The binuclear boron complexes containing two naphthyl groups are significantly red-shifted in comparison with the other binuclear boron-fluoride complexes. After the structures are characterized, these hemi-salen and salan ligand-based N, O-chelated binuclear boron-fluoride complexes were utilized to the transfer hydrogenation of the different acetophenone derivatives conversion to 1-phenylethanol derivatives as catalysts.     

The Reduction-Coupled Oxo Activation (ROA) Mechanism Responsible for the Catalytic Selective Activation and Functionalization of n-Butane to Maleic Anhydride by Vanadium Phosphate Oxide

We report here the results of density functional theory quantum mechanical (QM) studies of the detailed chemical mechanism underlying the n-butane selective oxidation to form maleic anhydride (MA) on vanadyl pyrophosphate [(VO)₂P₂O₇] and vanadyl phosphate [VOPO₄] surfaces. This QM-derived mechanism differs substantially from previous suggestions but is in excellent agreement with key experimental observations. We find that the O(1)=P bond of the oxidized X1 phase of the VOPO₄ surface is the active site for initiating the VPO chemistry, by extracting the H from the n-butane C–H bond. This contrasts sharply with previous suggestions, all of which involved the V=O bonds. The ability of O(1)=P to cleave alkane C–H bonds arises from a new unique mechanism that decouples the proton transfer and electron transfer components of this H atom transfer reaction. We find that the juxtaposition of a highly reducible V⁺⁵ next to the P=O bond but coupled via a bridging oxygen dramatically enhances the activity of the P=O bond to extract the proton from an alkane, while simultaneously transferring the electron to the V to form V⁺⁴. This Reduction-Coupled Oxo Activation (ROA) mechanism had not been known prior to these QM studies, but we believe that it may lead to a new strategy in designing selective catalysts for alkane activation and functionalization, and indeed it may be responsible for the selective oxidation by a number of known mixed metal oxide catalysts. To demonstrate the viability of this new ROA mechanism, we examine step by step the full sequence of reactions from n-butane to MA via two independent pathways. We that find that every step is plausible, with a highest reaction barrier of 21.7 kcal/mol.     

Green luminescence system containing a Tb3+‐β‐diketonate complex doped in the epoxy resin as sensitizer

Photoluminescent properties of the terbium tris(acetylacetonate)tetrahydrated [Tb(acac)₃(H₂O)₄], doped in the epoxy resin, in the solid state are reported. The polymeric Tb³⁺ system and the precursor terbium complex were characterized by elemental analysis, thermogravimetry, differential scanning calorimetry, and infrared and electronic spectroscopy. The excitation and emission spectra of the samples containing the Tb³⁺ complex doped diglycidyl epoxy resin were recorded at 298 and 77 K and exhibited the characteristic bands arising from the ⁵D₄ → ⁷F_J transitions (J = 6–0). The system shows an increase in the luminescence intensity with the increase in the Tb³⁺ complex in the 1, 5, and 10% concentrations due to the energy transfer from the polymer to the rare earth ion. On the other hand, the concentration quenching of luminescence of polymer doped with 15% of the Tb³⁺ complex was observed. The lifetime measurements (τ = 0.81, 0.80, 0.79, and 0.78 ms) decrease with the increase of Tb³⁺‐complex concentration (1, 5, 10, and 15%) doped in polymer. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 94: 865–870, 2004     

Inter-residual Hydrogen Bonding in Carbohydrates Unraveled by NMR Spectroscopy and Molecular Dynamics Simulations

Carbohydrates, also known as glycans in biological systems, are omnipresent in nature where they as glycoconjugates occur as oligo- and polysaccharides linked to lipids and proteins. Their three-dimensional structure is defined by two or three torsion angles at each glycosidic linkage. In addition, transglycosidic hydrogen bonding between sugar residues may be important. Herein we investigate the presence of these inter-residue interactions by NMR spectroscopy in D₂O/[D₆]DMSO (70:30) or D₂O and by molecular dynamics (MD) simulations with explicit water as solvent for disaccharides with structural elements α-d -Manp-(1→2)-d -Manp, β-d -GlcpNAc-(1→2)-d -Manp, and α-d -Glcp-(1→4)-β-d -Glcp, all of which have been suggested to exhibit inter-residue hydrogen bonding. For the disaccharide β-d -GlcpNAc-(1→2)-β-d -Manp-OMe, the large extent of O5′ ⋅⋅⋅ HO3 hydrogen bonding as seen from the MD simulation is implicitly supported by the ¹H NMR chemical shift and ³J_HO3,H3 value of the hydroxy proton. In the case of α-d -Glcp-(1→4)-β-d -Glcp-OMe, the existence of a transglycosidic hydrogen bond O2′⋅⋅⋅HO3 was proven by the presence of a cross-peak in ¹H,¹³C HSQC-TOCSY experiments as a result of direct TOCSY transfer between HO3 of the reducing end residue and H2′ (detected at C2′) of the terminal residue. The occurrence of inter-residue hydrogen bonding, albeit transient, is judged important for the stabilization of three-dimensional structures, which may be essential in maintaining a conformational state for carbohydrate–protein interactions of glycans to take place in biologically important environments.