Hyperfine-shifted 13C resonance assignments in an iron-sulfur protein with quantum chemical verification: aliphatic C-H···S 3-center-4-electron interactions

Although the majority of noncovalent interactions associated with hydrogen and heavy atoms in proteins and other biomolecules are classical hydrogen bonds between polar N-H or O-H moieties and O atoms or aromatic π electrons, high-resolution X-ray crystallographic models deposited in the Protein Data Bank show evidence for weaker C-H···O hydrogen bonds, including ones involving sp(3)-hybridized carbon atoms. Little evidence is available in proteins for the (even) weaker C-H···S interactions described in the crystallographic literature on small molecules. Here, we report experimental evidence and theoretical verification for the existence of nine aliphatic (sp(3)-hybridized) C-H···S 3-center-4-electron interactions in the protein Clostridium pasteurianum rubredoxin. Our evidence comes from the analysis of carbon-13 NMR chemical shifts assigned to atoms near the iron at the active site of this protein. We detected anomalous chemical shifts for these carbon-13 nuclei and explained their origin in terms of unpaired spin density from the iron atom being delocalized through interactions of the type: C-H···S-Fe, where S is the sulfur of one of the four cysteine side chains covalently bonded to the iron. These results suggest that polarized sulfur atoms in proteins can engage in multiple weak interactions with surrounding aliphatic groups. We analyze the strength and angular dependence of these interactions and conclude that they may contribute small, but significant, stabilization to the molecule.     

N-H...S hydrogen bonds in a ferredoxin model

The Fe4S4 complex {(CH3)3NCH2CONH2}2[Fe4S4((tBuS)4] (1) was synthesized to replicate the ferredoxin active site with a subset of its N-H...S hydrogen bonds. The two cationic counterions mimic the polypeptide backbone of ferredoxin (Fd) as amide hydrogen-bond donors to sulfur atoms of the iron-sulfur cluster. X-ray crystallographic data show that the organic sulfur (Sgamma) of one tert-butylthiolate ligand and one inorganic sulfur of the cluster core serve as N-H...S hydrogen-bond acceptors. The cluster core of complex 1 is tetragonally elongated in contrast to that of Fd, which is tetragonally compressed. This is the first observation of an elongated [Fe4S4]2+ cluster core. Additionally, this is the first synthetic Fd model in which N-H...S hydrogen bonding to a cluster has been achieved.     

Excited-state N-H···S hydrogen bond between indole and dimethyl sulfide: time-dependent density functional theory study

The intermolecular hydrogen bond N-H···S between indole and dimethyl sulfide is theoretically investigated. The formation of N-H···S hydrogen bonds between indole and dimethyl sulfide in ground and excited states is confirmed by the analysis of geometric structure, Mulliken charge, and infrared spectra. The result shows that the S(1) state of hydrogen bonded indole-Me(2)S is mainly a charger transfer state, while the S(2) state is a local excited state and also the state corresponding to the experiment. More importantly, it is demonstrated that the intermolecular hydrogen bond N-H···S of indole-Me(2)S is strengthened in the S(1) and S(2) states compared to that in ground state. Moreover, the strengthening of intermolecular N-H···S hydrogen bond in excited state induces the fluorescence emission peak of indole shifts to the red. These findings may provide insights for further study of N-H···S hydrogen bonds existing in many biomolecular systems.     

铁硫蛋白模型化合物的研究进展

本文综述了目前国际上对铁硫蛋白模型化合物研究的进展情况，介绍了NH---S氢键和芳香环在这些模型化合物及其天然蛋白中的作用。作为铁硫蛋白活性中心的模型，至今已有许多化合物被合成出来，通过这些模型化合物的研究知道，NH---S氢键和芳香环在调控配合物及天然蛋白的氧化还原电位和稳定性方面起着非常重要的作用。另外，作为顺式乌头酸酶、固氮酶等金属酶的模型，已成功地合成了含3Fe4S核以及含钼的铁硫簇合物。     

Inner-sphere reorganization energy of iron-sulfur clusters studied with theoretical methods

Models of several types of iron-sulfur clusters (e.g., Fe(4)S(4)(SCH(3))(4)(2-/3-/4-)) have been studied with the density functional B3LYP method and medium-sized basis sets. In a vacuum, the inner-sphere reorganization energies are 40, 76, 40, 62, 43, and 42 kJ/mol for the rubredoxin, [2Fe-2S] ferredoxin, Rieske, [4Fe-4S] ferredoxin, high-potential iron protein, and desulfoferrodoxin models, respectively. The first two types of clusters were also studied in the protein, where the reorganization energy was approximately halved. This change is caused by the numerous NH.S(Cys) hydrogen bonds to the negatively charged iron-sulfur cluster, giving rise to a polar local environment. The reorganization energy of the iron-sulfur clusters is low because the iron ions retain the same geometry and coordination number in both oxidation states. Cysteine ligands give approximately the same reorganization energy as imidazole, but they have the advantage of stabilizing a lower coordination number and giving more covalent bonds and therefore more effective electron-transfer paths.     

准确快速计算含多肽酰胺和核酸碱基的氢键复合物的氢键强度和氢键作用势能曲线

N-H···O=C、C-H···O=C、N-H···N和C-H···N等氢键作用是蛋白质a-螺旋结构、b-折叠结构和DNA双螺旋结构形成的主要因素,在生物分子识别、蛋白质复制以及遗传信息传递等过程中起重要作用。准确快速计算生物体系中存在的N-H···O=C、C-H···O=C、N-H···N和C-H···N等氢键作用强度以及氢键强度随分子几何结构(距离和角度)变化的势能曲线对正确模拟(从而正确认识和理解)蛋白质折叠机制和DNA双螺旋结构形成机制等生物过程意义重大,对设计合成具有特殊功能的生物分子材料有重要指导价值。本文主要介绍了近年来建立的偶极-偶极氢键作用模型及其在快速预测多肽-多肽分子间和核酸碱基-多肽分子间氢键作用强度和氢键作用势能曲线方面的应用。     

"Union is strength": how weak hydrogen bonds become stronger

Recently reported rotational spectroscopic studies on small dimers and oligomers bound by weak hydrogen bonds show that the driving forces, the spatial arrangement and the dynamical features displayed are very different from those involved in stronger and conventional hydrogen bonds. The very small binding energies (similar to those of van der Waals interactions) imply that the stabilization of the dimer is often obtained by networks of weak hydrogen bonds. Even in the presence of multiple bonds the partner molecules show a high degree of internal freedom within the complex. This paper analyses several examples of molecular adducts bound by weak hydrogen bonds formed in free jet expansions and recently characterized by rotational spectroscopy. They include weakly bound complexes of weak donors with strong acceptors (C-H···O,N, S-H···O,N), strong donors (O-H, N-H) with weak acceptors such as the halogen atoms and π systems but also the elusive interactions between weak donors and weak acceptors (C-H···π and C-H···halogen). Examples are also given where rotational spectroscopy highlights that weak hydrogen bonds are extremely important in chiral recognition phenomena and as driving forces of the conformational landscape of important biomolecules.     

Paramagnetic NMR spectroscopy and density functional calculations in the analysis of the geometric and electronic structures of iron-sulfur proteins

Paramagnetic NMR spectroscopy has been underutilized in the study of metalloproteins. One difficulty of the technique is that paramagnetic relaxation broadens signals from nuclei near paramagnetic centers. In systems with low electronic relaxation rates, this makes such signals difficult to observe or impossible to assign by traditional methods. We show how the challenges of detecting and assigning signals from nuclei near the metal center can be overcome through the combination of uniform and selective 2H, 13C, and 15N isotopic labeling with NMR experiments that utilize direct one-dimensional (2H, 13C, and 15N) and two-dimensional (13C-X) detection. We have developed methods for calculating NMR chemical shifts and relaxation rates by density functional theory (DFT) approaches. We use the correspondence between experimental NMR parameters and those calculated from structural models of iron-sulfur clusters derived from X-ray crystallography to validate the computational approach and to investigate how structural differences are manifested in these values. We have applied this strategy to three iron-sulfur proteins: Clostridium pasteurianum rubredoxin, Anabaena [2Fe-2S] ferredoxin, and human [2Fe-2S] ferredoxin. Provided that an accurate structural model of the iron-sulfur cluster and surrounding residues is available from diffraction data, our results show that DFT calculations can return NMR observables with excellent accuracy. This suggests that it might be possible to use calculations to refine structures or to generate structural models of active sites when crystal structures are unavailable. The approach has yielded insights into the electronic structures of these iron-sulfur proteins. In rubredoxin, the results show that substantial unpaired electron spin is delocalized across NH...S hydrogen bonds and that the reduction potential can be changed by 77 mV simply by altering the strength of one of these hydrogen bonds. In reduced [2Fe-2S] ferredoxins, hyperfine shift data have provided quantitative information on the degree of valence trapping. The approach described here for iron-sulfur proteins offers new avenues for detailed studies of these and other metalloprotein systems.     

The empirical correlation between hydrogen bonding strength and excited-state intramolecular proton transfer in 2-pyridyl pyrazoles

A series of 2-pyridyl pyrazoles 1a and 1-5 with various functional groups attached to either pyrazole or pyridyl moieties have been strategically designed and synthesized in an aim to probe the hydrogen bonding strength in the ground state versus dynamics of excited-state intramolecular proton transfer (ESIPT) reaction. The title compounds all possess a five-membered-ring (pyrazole)N-H···N(pyridine) intramolecular hydrogen bond, in which both the N-H bond and the electron density distribution of the pyridyl nitrogen lone-pair electrons are rather directional, so that the hydrogen bonding strength is relatively weak, which is sensitive to the perturbation of subtle chemical substitution and consequently reflected from the associated ESIPT dynamics. Various approaches such as (1)H NMR (N-H proton) to probe the hydrogen bonding strength and absorption titration to assess the acidity-basicity property were made for all the title analogues. The results, together with supplementary support provided by a computational approach, affirm that the increase of acidity (basicity) on the hydrogen bonding donor (acceptor) sites leads to an increase of hydrogen-bonding strength among the title 2-pyridyl pyrazoles. Luminescence results and the associated ESIPT dynamics further reveal an empirical correlation in that the increase of the hydrogen bonding strength leads to an increase of the rate of ESIPT for the title 2-pyridyl pyrazoles, demonstrating an interesting relationship among N-H acidity, hydrogen bonding strength, and the associated ESIPT rate.     

Conformational equilibrium and hydrogen bonding in liquid 2-phenylethylamine explored by Raman spectroscopy and theoretical calculations

2-Phenylethylamine (PEA) is the simplest aromatic amine neurotransmitter, as well as one of the most important. In this work, the conformational equilibrium and hydrogen bonding in liquid PEA were studied by means of Raman spectroscopy and theoretical calculations (DFT/MP2). By changing the orientation of the ethyl and the NH(2) group, nine possible conformers of PEA were found, including four degenerate conformers. Comparison of the experimental Raman spectra of liquid PEA and the calculated Raman spectra of the five typical conformers in selected regions (550-800 and 1250-1500 cm(-1)) revealed that the five conformers can coexist in conformational equilibrium in the liquid. The NH(2) stretching mode of the liquid is red-shifted by ca. 30 cm(-1) relative to that of an isolated PEA molecule (measured previously), implying that intermolecular N-H···N hydrogen bonds play an important role in liquid PEA. The relative intensity of the Raman band at 762 cm(-1) was found to increase with increasing temperature, indicating that the anti conformer might be favorable in liquid PEA at room temperature. The blue shift of the band for the bonded N-H stretch with increasing temperature also provides evidence of the existence of intermolecular N-H···N hydrogen bonds.     

Sulfur K-edge X-ray absorption spectroscopy of 2Fe-2S ferredoxin: covalency of the oxidized and reduced 2Fe forms and comparison to model complexes

Ligand K-edge X-ray absorption spectroscopy (XAS) provides a direct experimental probe of ligand-metal bonding. In previous studies, this method has been applied to mononuclear Fe-S and binuclear 2Fe-2S model compounds as well as to rubredoxins and the Rieske protein. These studies are now extended to the oxidized and reduced forms of ferredoxin I from spinach. Because of its high instability, the mixed-valence state was generated electrochemically in the protein matrix, and ligand K-edge absorption spectra were recorded using an XAS spectroelectrochemical cell. The experimental setup is described. The XAS edge data are analyzed to independently determine the covalencies of the iron-sulfide and -thiolate bonds. The results are compared with those obtained previously for the Rieske protein and for 2Fe-2S model compounds. It is found that the sulfide covalency is significantly lower in oxidized FdI compared to that of the oxidized model complex. This decrease is interpreted in terms of H bonding present in the protein, and its contribution to the reduction potential E degrees is estimated. Further, a significant increase in covalency for the Fe(III)-sulfide bond and a decrease of the Fe(II)-sulfide bond are observed in the reduced Fe(III)Fe(II) mixed-valence species compared to those of the Fe(III)Fe(III) homovalent site. This demonstrates that, upon reduction, the sulfide interactions with the ferrous site decrease, allowing greater charge donation to the remaining ferric center. That is the dominant change in electronic structure of the Fe(2)S(2)RS(4) center upon reduction and can contribute to the redox properties of this active site.     

Dynamics of the intermolecular hydrogen bonds in the polymorphs of paracetamol in relation to crystal packing and conformational transitions: a variable-temperature polarized Raman spectroscopy study

The properties of the intermolecular hydrogen bonds in the monoclinic (Form I) and the orthorhombic (Form II) polymorphs of paracetamol, C(8)H(9)NO(2), have been studied by single crystal polarized Raman spectroscopy (40 to 3700 cm(-1)) in a wide temperature range (5 K < T < 300 K) in relation to the dynamics of methyl-groups of the two forms. A detailed analysis of the temperature dependence of the wavenumbers, bandwidths and integral intensities of the spectral bands has revealed an essential difference between the two polymorphs in the strength and ordering of OH···O and NH···O hydrogen bonds. The compression of intermolecular hydrogen bonds is interrelated with crystal packing and the dynamics of methyl-groups. On structural compression of the orthorhombic polymorph on cooling, a compromise is to be sought between the shortening of OH···O and NH···O bonds, attractive CH···O and repulsive CH···H contacts in the crystal structure. As a result of a steric conflict at temperatures below 100 K, N-H···O hydrogen bonds become significantly disordered, and an extended intramolecular transition from the conformation "staggered" with respect to the C=O bond to the one "staggered" with respect to the NH bond is observed. In most of the studied crystals this transition was only about 60% complete even at 5 K, but in some of the crystals the orientation of all the methyl-groups became staggered with respect to the NH bond at low temperatures. This complete transition was coupled to a sharp shortening of the OH···O and NH···O hydrogen bonds at <100 K, the appearance of new additional positions of the protons in these H-bonds, and a slight strengthening of the C-HO bonds formed by methyl-groups. The same conformational transition has been observed also in the monoclinic polymorph at T < 80 K. The crystal packing in Form I prevents the O-H···O hydrogen bonds from adopting the optimum geometry, and they are significantly disordered at all the temperatures, especially at ≤200 K. The packing of molecules in Form I is also not favourable to form C-H···O hydrogen bonds involving methyl-groups. One can conclude from the comparison of diffraction and spectroscopic data that the higher stability of Form I results not from a larger strength of individual OH···O and NH···O hydrogen bonds, but is a cumulative effect: all the hydrogen bonds together stabilize the structure of the monoclinic polymorph more than that of the orthorhombic polymorph.     

Moderate and advanced intramolecular proton transfer in urea-anion hydrogen-bonded complexes

The study of the interactions of the three urea-based receptors AH, BH(+) and CH(2+) with a variety of anions, in MeCN, has made it possible to verify the current view that hydrogen bonding is frozen proton transfer from the donor (the urea N-H fragment in this case) to the acceptor (the anion X(-)). The poorly acidic, neutral receptor AH establishes two equivalent hydrogen bonds N-H···X(-), with all anions, including CH(3)COO(-) and F(-), in which moderate proton transfer from N-H to the anion takes place. The strongly acidic, dicationic receptor CH(2+) forms, with most anions, complexes in which two inequivalent hydrogen bonds are present: one involving moderate proton transfer (N-H···X(-)) and one in which advanced proton transfer has taken place, described as N(-)···H-X. The degree of proton advancement is directly related to the basic tendencies of the anion. The cationic receptor BH(+) of intermediate acidic properties only forms complexes with two inequivalent hydrogen bonds (moderate+advanced proton transfer) with CH(3)COO(-) and F(-), and complexes with two equivalent hydrogen bonds (moderate proton transfer) with all the other anions. Moreover, [B···HF] and [C···HF](+), on addition of a second F(-) ion, lose the bound HF molecule to give HF(2)(-). Release of CH(3)COOH, with the formation of [CH(3)COOH···CH(3)COO](-), also takes place with the [B···CH(3)COOH] complex in the presence of a large excess of anion.     

Ab initio study of ternary complexes X:(HCNH)(+):Z with X, Z = NCH, CNH, FH, ClH, and FCl: diminutive cooperative effects on structures, binding energies, and spin-spin coupling constants across hydrogen bonds

Ab initio calculations have been performed on a series of complexes in which (HCNH)(+) is the proton donor and CNH, NCH, FH, ClH, and FCl (molecules X and Z) are the proton acceptors in binary complexes X:HCNH(+) and HCNH(+):Z, and ternary complexes X:HCNH(+):Z. These complexes are stabilized by C-H(+)···A and N-H(+)···A hydrogen bonds, where A is the electron-pair donor atom of molecules X and Z. Binding energies of the ternary complexes are less than the sum of the binding energies of the corresponding binary complexes. In general, as the binding energy of the binary complex increases, the diminutive cooperative effect increases. The structures of these complexes, data from the AIM analyses, and coupling constants (1)J(N-H), (1h)J(H-A), and (2h)J(N-A) for the N-H(+)···A hydrogen bonds, and (1)J(C-H), (1h)J(H-A), and (2h)J(C-A) for the C-H(+)···A hydrogen bonds provide convincing evidence of diminutive cooperative effects in these ternary complexes. In particular, the symmetric N···H(+)···N hydrogen bond in HCNH(+):NCH looses proton-shared character in the ternary complexes X:HCNH(+):NCH, while the proton-shared character of the C···H(+)···C hydrogen bond in HNC:HCNH(+) decreases in the ternary complexes HNC:HCNH(+):Z and eventually becomes a traditional hydrogen bond as the strength of the HCNH(+)···Z interaction increases.     

Structure of 7-azaindole···2-fluoropyridine dimer in a supersonic jet: competition between N-H···N and N-H···F interactions

In the present work, we have investigated the structure of 7-azaindole···2-fluoropyridine dimer in a supersonic jet by employing resonant two photon ionization (R2PI), IR-UV, and UV-UV double resonance spectroscopic techniques combined with quantum chemistry calculations. The R2PI spectrum of the dimer is recorded by electronic excitation of the 7-azaindole moiety, and a few low frequency intermolecular vibrations of the dimer are clearly observed in the spectrum. The electronic origin band of the dimer is red-shifted by 1278 cm(-1) from the S(1) ← S(0) origin band of 7-azaindole monomer. The presence of a single conformer of the dimer is confirmed by IR-UV and UV-UV hole-burning spectroscopic techniques. RIDIR (Resonant ion dip infrared) spectrum of the dimer shows a red-shift of 265 cm(-1) in the N-H stretching frequency with respect to that of the 7-azaindole monomer. Two planar double hydrogen bonded cyclic structures of the dimer have been predicted from DFT calculations. Comparison of experimental and theoretical N-H stretching frequencies confirms that the observed dimer is stabilized by N-H···N and C-H···N hydrogen bonding interactions. The less stable conformer with N-H···F and C-H···N interactions are not observed in the experiment. The competition between N-H···N and N-H···F interactions in the two dimeric structures are discussed from natural bond orbital (NBO) analysis. The current results demonstrate that fluorine makes a hydrogen bond of intermediate strength through cooperative interaction of another hydrogen bond (C-H···N) present in the dimer, although fluorine is believed to be very weak hydrogen bond acceptor.     

Photophysical, electrochemical and anion sensing properties of Ru(II) bipyridine complexes with 2,2'-biimidazole-like ligand

A new anion sensor [Ru(bpy)(2)(DMBbimH(2))](PF(6))(2) (3) (bpy is 2, 2'-bipyridine and DMBbimH(2) is 7,7'-dimethyl-2,2'-bibenzimidazole) has been developed. Its photophysical, electrochemical and anion sensing properties are compared with two previously investigated systems, [Ru(bpy)(2)(BiimH(2))](PF(6))(2) (1) and [Ru(bpy)(2)(BbimH(2))](PF(6))(2) (2) (BiimH(2) is 2,2'-biimidazole and BbimH(2) is 2,2'-bibenzimidazole). The high acidity of the N-H fragments in these complexes make them easy to be deprotonated by strong basic anions such as F(-) and OAc(-), and they form N-H···X hydrogen bonds with weak basic anions like Cl(-), Br(-), I(-), NO(3)(-), and HSO(4)(-). Complex 3 displays strong hydrogen bonding with these 5 weak basic anions, with binding constants between 17,000 and 21,000, which are larger than those observed in complex 1, with binding constants between 3300 and 5700, and in complex 2, which shows no hydrogen bonding toward Cl(-), Br(-), I(-), and NO(3)(-), and forms considerable hydrogen bonds with HSO(4)(-) with a binding constant of 11,209. These hydrogen bonding behaviours give different NMR, emission and electrochemical responses. The different anion binding affinity of these complexes may be mainly attributed to their different pK(a1) values, 7.2 for 1, 5.7 for 2, and 6.2 for 3. The additional methyl groups at the 7 and 7' positions of complex 3 may also play an important role in the enhancement of anion binding strength.     

N-methylformamide-benzene complex as a prototypical peptide N-H...pi hydrogen-bonded system: density functional theory and MP2 studies

Although the existence of peptide N-H...pi hydrogen bonds recently has been reported in protein structures, little is known about their strength and binding nature and, therefore, the relative importance of the interaction. To shed light on this binding, the N-methylformamide-benzene complex, as a model of the peptide N-H...pi hydrogen bonding, was studied by using density functional theory and M?ller-Plesset second-order perturbation (MP2) methods. The geometry of the complex was fully optimized at the B3LYP/6-31G(d,p) and MP2/6-31G(d,p) levels. The optimized interaction distances are about 3.6 and 3.2 A, respectively, at the two levels. The binding energy corrected by basis set superposition error with the MP2/cc-pVTZ method based on the MP2/6-31G geometry is -4.37 kcal/mol, which is as strong as the conventional hydrogen bonding. The calculated results suggest that the peptide N-H...pi hydrogen bonding is of sufficient strength to play an important role in the stabilization of protein structures. These results are helpful to better understand the characteristics and nature of the peptide N-H...pi interaction as well as to modify current force fields to better represent this special interaction.     

4-(4-吡啶基)嘧啶-2-磺酸钠与过渡金属组装的三维氢键网络结构

4-(4-吡啶基)嘧啶-2-磺酸钠(NaL²)与金属盐(M=Mn,Zn,Co,Fe)组装分别得到4个配合物,其分子式为[ML₂·4H₂O]·2H₂O。氢键在这些化合物的超分子结构中起了重要作用。4种配合物中磺酸基的氧原子没有螯合金属离子,而是作为氢键的受体,同时水分子作为氢键给体和受体起到双重作用。另外,在配合物2～4中,杂环上的碳原子也起到供体的作用与磺酸基的氧原子形成氢键。     

Highly covalent ferric-thiolate bonds exhibit surprisingly low mechanical stability

Depending on their nature, different chemical bonds show vastly different stability with covalent bonds being the most stable ones that rupture at forces above nanonewton. Studies have revealed that ferric-thiolate bonds are highly covalent and are conceived to be of high mechanical stability. Here, we used single molecule force spectroscopy techniques to directly determine the mechanical strength of such highly covalent ferric-thiolate bonds in rubredoxin. We observed that the ferric-thiolate bond ruptures at surprisingly low forces of ～200 pN, significantly lower than that of typical covalent bonds, such as C-Si, S-S, and Au-thiolate bonds, which typically ruptures at >1.5 nN. And the mechanical strength of Fe-thiolate bonds is observed to correlate with the covalency of the bonds. Our results indicated that highly covalent Fe-thiolate bonds are mechanically labile and display features that clearly distinguish themselves from typical covalent bonds. Our study not only opens new avenues to investigating this important class of chemical bonds, but may also shed new lights on our understanding of the chemical nature of these metal thiolate bonds.     

The strength of hydrogen bonding to metal-bound ligands can contribute to changes in the redox behaviour of metal centres

A series of nine tripodal tetradentate ligands based on tris(pyridyl-2-methyl)amine TPA with hydrogen bond donors R in one, two and three of the pyridine 6-positions (R = NH2 amino, L(Am-1,2,3); NHCH2(t)Bu neopentylamino, L(Np-1,2,3); NHCO(t)Bu pivaloylamido, L(Piv-1,2,3)) and TPA are used to investigate the effect of different hydrogen bonding microenvironments on electrochemical properties of their LCuCl complexes. The hydrogen bond donors are rigidly preorganised and suitably oriented for intramolecular N-H...Cl-Cu hydrogen bonds. Cyclic voltammetry studies show that the reduction potential of the Cu(II)/Cu(I) couple as a function of the ligand follows the order TPA < L(Am-n) < or approximately L(Np-n) < L(Piv-n), and that the magnitude of the effect increases with the number of hydrogen bonding groups. These trends could be explained in terms of the steric and electronic effects exerted by these groups stabilising the Cu(I) oxidation state. In fact, the X-ray structure of the air-stable [(L(Piv-3))Cu(I)Cl] complex is reported and shows elongated Cu-N and Cu-Cl bonds, presumably due to the combination of steric and electron withdrawing effects exerted by the three pivaloylamido groups. We reasoned that the strength of hydrogen bonding in the Cu(I) and Cu(II) oxidation states could differ and therefore contribute also to the aforementioned redox changes; this hypothesis is tested using IR and NMR spectroscopy. IR studies of the [(L(Piv-1,2,3))Cu(I)Cl] and [(L(Piv-1,2,3))Cu(II)Cl]+ complexes in acetonitrile show that the intramolecular N-H...Cl-Cu hydrogen bonding weakens in the order L(Piv-1) > L(Piv-2) > L(Piv-3), and that it is stronger in the Cu(I) complexes. The 1H NMR spectra of the [(L(Piv1,2,3))Cu(I)Cl] complexes are in complete agreement with the IR data, and reveal that the stability of the Cu(I) complexes to oxidation in air increases in the order L(Piv-1) < L(Piv-2) < L(Piv-3). The hydrogen bonds in the Cu(I) complexes are stronger because of the higher electron density on the Cl ligand, when compared to the Cu(II) complexes. This is consistent with ab initio MP2 calculations performed on the complexes [(L(Piv-3))Cu(I)Cl] and [(L(Piv-3))Cu(II)Cl]+. Thus, the electron density of a metal-bound ligand acting as hydrogen bond acceptor is revealed as the major factor in determining the strength of the hydrogen bonds formed. From the IR data the energies of the N-H...Cl-Cu hydrogen bonds is estimated, as is the contribution of changes in hydrogen bond strength with the oxidation state of the copper centre and number of interactions to stabilising the Cu(I) state. Some of the implications of this result in dioxygen activation chemistry are discussed.